SCHIZOPHRENIA-RELATED MICRODELETION GENE 2510002D24Rik IS ESSENTIAL FOR SOCIAL MEMORY

ABSTRACT

Described is a method for treating a social memory deficit in a subject with a neuropsychiatric disease is treated by administering a peptide encoded by 2510002D24Rik or Atp23 genes, a vector expressing such peptide, or an agent capable of increasing the level or activity of such peptide. The method may be used to treat schizophrenia or autism. The peptide, vector or agent can be administered directly to the hippocampus, such as by transcranial surgical injection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/889,111, filed Aug. 20, 2019, the disclosure of which is hereinincorporated by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under MH097742 awardedby the National Institutes of Health. The government has certain rightsin the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 6, 2020, isnamed 243734_000139_SL.txt and is 17,315 bytes in size.

FIELD

This application relates to compositions for use in treatingschizophrenia spectrum disorders, autism spectrum disorder, and anyother disorder or condition arising from a social memory deficit. Theapplication further relates to methods of treating schizophreniaspectrum disorders, autism spectrum disorder, and any other disorder orcondition arising from a social memory deficit.

BACKGROUND

The 22q11.2 deletion syndrome (22q11DS) is associated with high risk ofdeveloping schizophrenia symptoms, including psychosis, later in life.22q11DS is a leading genetic cause of schizophrenia and instigated bythe hemizygous deletion of multiple genes (1.5-3 Mb) of the q (long) armof chromosome 22 in humans. In particular, 22q11DS carries a 25- to30-fold risk of schizophrenia.⁹⁻¹¹

Schizophrenia may cause specific changes in area CA2, a long over-lookedregion of the hippocampus recently found to be critical for socialmemory formation. Silencing or lesioning the CA2 area of the murinehippocampus is known to cause a specific deficit in social-recognitionmemory, with no change in sociability or spatial and contextual memoriestypically associated with the hippocampus.^(1,5)

SUMMARY OF THE INVENTION

There is a great need in the art to develop effective treatments for22q11DS, including treatment of positive symptoms of mental disordersand schizophrenia, such as, e.g., hallucinations, delusions,disorganized thought, and psychosis. The present invention addressesthis and other needs.

In one aspect is provided a method for treating a social memory deficitin a subject with a neuropsychiatric disease, the method comprisingadministering to the subject a therapeutically effective amount of (i) aprotein encoded by a 2510002D24Rik gene or a functional derivative orfragment thereof, or (ii) a vector expressing said protein encoded bysaid 2510002D24Rik gene or a functional derivative or fragment thereof.

In another aspect is provided a method for treating a social memorydeficit in a subject with a neuropsychiatric disease, said methodcomprising administering to the subject a therapeutically effectiveamount of (i) a protein encoded by an Atp23 gene or a functionalderivative or fragment thereof, or (ii) a vector expressing said proteinencoded by said Atp23 gene or a functional derivative or fragmentthereof.

In some embodiments of the above aspects, the administration results inreplenishing Atp23 level in CA2 interneurons of the subject. In aspecific embodiment, the CA2 interneurons are parvalbumin (PV)-positiveinterneurons.

In various embodiments, the administration results in replenishing Atp23level in CA2 area of the hippocampus of the subject. In someembodiments, replenishing the Atp23 level is to the level found inhealthy subjects.

In some embodiments, the neuropsychiatric disease is selected fromschizophrenia spectrum disorders, 22q11 deletion syndrome, and autismspectrum disorders. In some embodiments, the vector is selected fromadeno-associated virus (AAV) vectors, retrovirus vectors, adenovirusvectors, Sindbis virus vectors, vaccinia virus vectors, and herpes virusvectors. In a specific embodiment, the vector is an AAV vector. In someembodiments, the AAV vector has a capsid from a serotype selected fromAAV1, AAV2, AAV5, AAV8, and AAV9. In certain embodiments, the retrovirusvector is a lentivirus vector.

In various embodiments, the expression of the protein or functionalderivative or fragment thereof in the vector is controlled by a promoterselected from the group consisting of fsst promoter, hDlx promoter, mDlxpromoter, Synapsin promoter, CMV promoter, β-actin promoter, and CamKIIapromoter. In some embodiments, the expression of the protein orfunctional derivative or fragment thereof in the vector is controlled bya pan-GABAergic interneuron promoter.

In some embodiments, the administration is via injection into the CA2area of the hippocampus of the subject. In some embodiments, theadministration is via a transcranial surgical injection. In specificembodiments, the administration is systemic. In specific embodiments,the administration is intranasal.

In some embodiments, the protein encoded by 2510002D24Rik gene comprisesthe amino acid sequence which has at least 80% sequence identity to SEQID NO: 1. In some embodiments, the protein encoded by 2510002D24Rik genecomprises the amino acid sequence which has at least 90% sequenceidentity to SEQ ID NO: 1. In some embodiments, the protein encoded by2510002D24Rik gene comprises the amino acid sequence SEQ ID NO: 1. Insome embodiments, the protein encoded by 2510002D24Rik gene consists ofthe amino acid sequence SEQ ID NO: 1.

In some embodiments, the protein encoded by Atp23 gene comprises theamino acid sequence which has at least 80% sequence identity to SEQ IDNO: 2. In some embodiments, the protein encoded by Atp23 gene comprisesthe amino acid sequence which has at least 90% sequence identity to SEQID NO: 2. In some embodiments, the protein encoded by Atp23 genecomprises the amino acid sequence SEQ ID NO: 2. In some embodiments, theprotein encoded by Atp23 gene consists of the amino acid sequence SEQ IDNO: 2.

In various embodiments, the subject is human.

In another aspect is provided a pharmaceutical composition comprising aprotein encoded by 2510002D24Rik gene or a functional derivative orfragment thereof and a pharmaceutically acceptable carrier or excipient.

In another aspect is provided a pharmaceutical composition comprising avector encoding a protein encoded by 2510002D24Rik gene or a functionalderivative or fragment thereof and a pharmaceutically acceptable carrieror excipient. In some embodiments, the vector is selected fromadeno-associated virus (AAV) vectors, retrovirus vectors, adenovirusvectors, Sindbis virus vectors, vaccinia virus vectors, and herpes virusvectors. In certain embodiments, the vector is an AAV vector. In someembodiments, the AAV vector has a capsid from a serotype selected fromAAV1, AAV2, AAV5, AAV8, and AAV9. In certain embodiments, the retrovirusvector is a lentivirus vector.

In some embodiments, in the vector the sequence encoding the proteinencoded by 2510002D24Rik gene or functional derivative or fragmentthereof is operably linked to a promoter selected from the groupconsisting of fsst promoter, hDlx promoter, mDlx promoter, Synapsinpromoter, CMV promoter, β-actin promoter, and CamKIIa promoter. In someembodiments, in the vector the sequence encoding the protein encoded by2510002D24Rik gene or functional derivative or fragment thereof isoperably linked to a pan-GABAergic interneuron promoter. In someembodiments, the protein encoded by 2510002D24Rik gene comprises theamino acid sequence which has at least 90% sequence identity to SEQ IDNO: 1. In some embodiments, the protein encoded by 2510002D24Rik genecomprises the amino acid sequence SEQ ID NO: 1. In some embodiments, theprotein encoded by 2510002D24Rik gene consists of the amino acidsequence SEQ ID NO: 1.

In another aspect is provided a pharmaceutical composition comprising aprotein encoded by Atp23 gene or a functional derivative or fragmentthereof and a pharmaceutically acceptable carrier or excipient.

In another aspect is provided a pharmaceutical composition comprising avector encoding a protein encoded by Atp23 gene or a functionalderivative or fragment thereof and a pharmaceutically acceptable carrieror excipient. In some embodiments, the vector is selected fromadeno-associated virus (AAV) vectors, retrovirus vectors, adenovirusvectors, Sindbis virus vectors, vaccinia virus vectors, and herpes virusvectors. In certain embodiments, the vector is an AAV vector. In someembodiments, the AAV vector has a capsid from a serotype selected fromAAV1, AAV2, AAV5, AAV8, and AAV9. In certain embodiments, the retrovirusvector is a lentivirus vector.

In various embodiments, in the vector the sequence encoding the proteinencoded by Atp23 gene or functional derivative or fragment thereof isoperably linked to a promoter selected from the group consisting of fsstpromoter, hDlx promoter, mDlx promoter, Synapsin promoter, CMV promoter,β-actin promoter, and CamKIIa promoter. In some embodiments, in thevector the sequence encoding the protein encoded by Atp23 gene orfunctional derivative or fragment thereof is operably linked to apan-GABAergic interneuron promoter. In some embodiments, the proteinencoded by Atp23 gene comprises the amino acid sequence which has atleast 90% sequence identity to SEQ ID NO: 2.

In some embodiments, the protein encoded by Atp23 gene comprises theamino acid sequence SEQ ID NO: 2. In some embodiments, the proteinencoded by Atp23 gene consists of the amino acid sequence SEQ ID NO: 2.

In various embodiments, the pharmaceutical composition is formulated forinjection into hippocampus. In various embodiments, the pharmaceuticalcomposition is formulated for transcranial surgical injection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H show the results of experiments on deficits in socialmemory, CA2 fast-spiking interneuron firing, long-term depression atCA3-CA2 inhibitory synapses, and disinhibitory plasticity in CA2pyramidal neurons of 2510002D24Rik-deficient mice. In FIGS. 1A and 1B,the left panel shows direct interaction one-chamber test using the same(FIG. 1A) or a different (FIG. 1B) stimulus animal in the 2 trials. InFIG. 1A, the right panel shows that only WT mice displayed decreasedinvestigation during trial 2 of the mouse encountered in trial 1 (WT,n=13, *P=002; Rik^(+/−), n=19, P=0.331; Rik^(−/−), n=17, P=0.113).Two-way repeated measures (RM) analysis of variance (ANOVA):F(1,46)=12.133, *P=0.001; trial 1: all pair-wise comparisons P>0.05;trial 2: WT vs. Rik^(+/−), *P=0.032, WT vs. Rik^(−/−), *P=0.026. In FIG.1B, mutant and WT mice explored the 2 different stimulus animalssimilarly (two-way RM ANOVA: F(1,46)=2.893, P=0.096). FIGS. 1C and 1Dshow input-output curves of the compound EPSP/IPSP (PSP) amplitudes incontrol conditions (FIG. 1C) and after blocking inhibition with SR95531(1 μm) and CGP55845 (1 μm) (FIG. 1D) in response to Schaffer collateralstimulation in WT (c: n=16 neurons in 6 mice; d: 12 neurons, 6 mice),Rik^(+/−) (c: 16 neurons, 6 mice; d: 9 neurons, 4 mice), and Rik^(−/−)(c: 20 neurons, 5 mice; d: 13 neurons, 6 mice) mice. In FIG. 1C, theparameters for RM ANOVA are: F(2;49)=5.998, genotype *P=0.005. In FIG.1D, the parameters for two-way RM ANOVA are: F(2;31)=0.563, genotypeP=0.574). Insets show representative compound PSP traces. FIG. 1E showsmean IPSCs recorded from CA2 pyramidal neurons in response tostimulation of Schaffer collaterals in WT (13 neurons, 3 mice),Rik^(+/−) (9 neurons, 3 mice), and Rik^(−/−) (10 neurons; 2 mice)animals. In FIG. 1E, the parameters for two-way RM ANOVA are:F(2,29)=6.755, genotype *P=0.004. In FIG. 1F, examples (left) and meannumbers (right) of APs fired in response to 150-pA (1 s) depolarizationof CA2 interneurons of WT (10 neurons, 4 mice), Rik^(+/−) (12 neurons, 4mice), and Rik^(−/−) (10 neurons; 4 mice) animals. The parameters forKruskal-Wallis one-way ANOVA on ranks are: H=9.643; genotype *P=0.008(WT vs Rik^(+/−) and WT vs Rik^(−/−): *P<0.05). FIG. 1G shows meannormalized IPSCs recorded in CA2 pyramidal neurons in the voltage-clampconfiguration, before and after tetanic stimulation of Schaffercollaterals (100 pulses at 100 Hz twice, arrows) in WT (8 neurons, 5mice), Rik^(+/−) (12 neurons, 7 mice), and Rik^(−/−) (15 neurons; 7mice) animals. The parameters for one-way ANOVA are: F(2,32)=6.044,genotype P=0.006 (WT vs. Rik^(+/−), *P=0.006; WT vs. Rik^(−/−),*P=0.018). FIG. 1H shows mean normalized PSP recorded in CA2 pyramidalneurons in the current-clamp configuration, before and after tetanicstimulation of Schaffer collaterals (arrows) in WT (7 neurons, 4 mice),Rik^(+/−) (6 neurons, 3 mice), and Rik^(−/−) (6 neurons; 3 mice)animals. The parameters for one-way ANOVA are: F(2,16)=5.662, genotypeP=0.014 (WT vs. Rik^(+/−), *P=0.026; WT vs. Rik^(−/−), *P=0.027). InFIGS. 1G and 1H, the insets show representative traces of IPSPs (FIG.1G) and PSPs (FIG. 1H) before (dark traces) and after (light traces)tetanic stimulation.

FIGS. 2A-2H show the results of experiments demonstrating thatdeficiency of the 2510002D24Rik-encoded product, which interacts with amitochondrial protein Atp23, reduces ATP-ADP interconversion in CA2interneurons. FIGS. 2A and 2B show the results of a tandem mass tag(TMT) mass spectrometry-based proteomics analysis in whole-hippocampaltissue (FIG. 2A) and hippocampal synaptosomes (FIG. 2B) from WT andRik^(−/−) mice revealed 3 hits encoded by 2510002D24Rik and Atp23 genes(red) that are substantially reduced in mutants. In FIG. 2C, Westernblotting confirmed the reduction of Atp23 in the whole-hippocampallysate of Rik^(−/−) mice. FIG. 2D shows that Atp23 co-precipitates with2510002D24Rik^(HA) fusion protein (HA) in the hippocampal lysates fromRik^(HA) mice. The Atp23 signal was stronger in the HA-pulledimmunoprecipitate (IP) than in the input lysate or the flow-through (FT)fraction that remained after immunoprecipitation. FIG. 2E showsenrichment of 2510002D24Rik^(HA) fusion protein (HA) and Atp23 inmitochondrial fractions (M) compared to synaptosomal fractions (S)prepared from the hippocampus of Rik^(HA) mice. FIG. 2F shows examples(top) and averages (bottom) of PercevalHR fluorescence excited at 840 nm(ADP, upward sweeps) or 940 nm (ATP, downward sweeps) before, during,and after 300-pA depolarization measured through line scans in cellbodies of CA1 interneurons of WT and 2510002D24Rik-deficient mice. InFIGS. 2G and 2H, the mean area under curves (AUC) for 840-nm-excited(FIG. 2G) and 940-nm-excited (FIG. 2H) PercevalHR fluorescence in WT(n=7); Rik^(/−) (n=7), and Rik^(−/−) (n=7) neurons. In FIG. 2G, theparameters for one-way ANOVA are: F(2;18)=4.251, genotype *P=0.031 (WTvs. Rik^(+/−), *P=0.033, WT vs. Rik^(−/−), *P=0.027). In FIG. 2H, theparameters for one-way ANOVA are: one-way ANOVA: F(2,18)=7.9, genotype*P=0.003 (WT vs. Rik^(−/−), *P=0.003, WT vs. Rik^(−/−), *P=0.007).

FIGS. 3A-3H show that Atp23 deletion or 2510002D24Rik conditionaldeletion causes deficits in firing and synaptic plasticity in CA2interneurons and social memory. FIG. 3A shows examples of firing (left)and mean numbers of APs (right) induced by 150-pA (1 s) depolarizationin CA2 interneurons of WT (14 neurons, 3 mice) and Atp23^(+/−) (17neurons, 3 mice) animals. Mann-Whitney Rank-Sum test: U=56.5, *P=0.014.FIG. 3B shows average normalized IPSCs measured before and after tetanicstimulation (arrows) of Schaffer collaterals in WT (12 neurons, 7 mice)and Atp23^(−/−) (7 neurons, 4 mice) CA2 pyramidal neurons. Two-tailedt-test t=−2.428, *P=0.028. FIGS. 3C and 3D show the results of a directinteraction one-chamber test using the same (FIG. 3C) or different (FIG.3D) stimulus animal in the 2 trials in WT (n=12) and Atp23^(+/−) (n=11)mice. In FIG. 3C, there was a significant difference between the groups.Two-way RM ANOVA: trial F(1,21)=11.621, *P=0.003. WT (*P<0.001) but notAtp23^(+/−) (*P=0.374) mice showed less investigation during trial 2than in trial 1. There was no difference between genotypes in trials 1(P=0.062) or 2 (P=0.816). FIG. 3D shows the results of an experiment inwhich WT and Atp23^(+/−) mice explored the 2 different stimulus animalssimilarly. Two-way RM ANOVA: F(1,21)=2.757, P=0.112. FIG. 3E showsexamples of firing (left) and mean numbers of APs (right) induced by150-pA (1 s) depolarization in labelled CA2 interneurons of WT (11neurons, 3 mice) and Rik^(f/f) mice injected with AAV-hDlx-cre-GFP.Rik^(f/f);CA2^(AVV-hDlx-cre) (n=10 neurons, 3 mice) neurons firedsignificantly fewer APs than did WT;CA2^(AVV-hDlx-cre) neurons (n=11neurons, 3 mice). Mann-Whitney Rank-Sum test U=19.5, *P=0.014. In FIG.3F, the average normalized IPSCs were measured before and after tetanicstimulation (arrows) of Schaffer collaterals in WT;CA2^(AVV-hDlx-cre) (6neurons, 3 mice) and Rik^(f/f); CA2^(AVV-hDlx-cre) (6 neurons, 4 mice)CA2 pyramidal neurons. Two-tailed t-test: t=−2.804, *P=0.019. FIG. 3Gshows the results of a direct interaction one-chamber test using thesame stimulus animal in the 2 trials revealed that WT;CA2^(AVV-hDlx-cre)(n=5, *P=0.027) mice but not Rik^(f/f); CA2^(AVV-hDlx-cre) (n=6,P=0.616) mice showed less investigation between the trials. Two-way RMANOVA: F(1,9)=6.01, *P=0.037. In FIG. 3H, a direct interactionone-chamber test using different stimulus animals in the 2 trialsrevealed no difference between WT;CA2^(AVV-hDlx-cre) (n=4) andRik;CA2^(AVV-hDlx-cre) (n=5) mice. Two-way RM ANOVA: F(1,7)=1.342,P=0.361. Insets (FIGS. 38 and 3F) show representative traces of IPSCsbefore (solid lines) and after (dash lines) tetanic stimulation.

FIGS. 4A-4F show that replenishing Atp23 in CA2 interneurons rescuesdeficiencies in interneuron firing, CA3-CA2 synaptic plasticity, andsocial memory in 2510002D24Rik-deficient mice. FIG. 4A is an image ofthe hippocampus infected with AAV-fsst-Atp23-GFP (green) in the CA2area, which is marked by RGS14 (red). The scale bar represents 200 μm.FIG. 4B shows examples and mean numbers of APs in labelled CA2interneurons infected with AAV-fsst-RFP [CA2^(AVV-fsst-RFP) (RFP)] orAAV-fsst-Atp23-GFP [CA2^(AVV-fsst-Atp23) (Atp23-GFP)] in WT (11 neurons,6 neurons), Rik^(+/−) (9 neurons, 10 neurons), and Rik^(−/−) (8 neurons,11 neurons) mice. RFP: one-way ANOVA, F(2,24)=4.737, *P=0.018. WT vs.Rik^(+/−) (*P=0.035), WT vs Rik^(−/−) (*P=0.037). Atp23-GFP: one-wayANOVA, F(2,24)=0.215, P=0.808. FIG. 4C shows average normalized IPSCsmeasured before and after tetanic stimulation (arrows) of Schaffercollaterals in CA2 pyramidal neurons of WT (n=10, n=6), Rik^(+/−) (8neurons, 6 neurons) Rik^(−/−) mice (11 neurons, 7 neurons) injected withAAV-fsst-RFP (RFP, left) or AAV-fsst-Atp23-GFP (Atp23-GFP, right). RFP:one-way ANOVA, F(2,25)=7.792, *P=0.002. WT vs Rik^(+/−), *P=0.003, WTvs. Rik^(−/−), *P=0.037. Atp23-GFP: one-way ANOVA, F(2,16)=2.313,P=0.131. FIG. 4D shows mean normalized PSP recorded in CA2 pyramidalneurons in the current-clamp configuration, before and after delivery oftetanic stimulation of Schaffer collaterals (arrows) in WT (9 neurons, 8neurons), Rik^(+/−) (9 neurons, 7 neurons), and Rik^(−/−) (7 neurons, 6neurons) animals injected with AAV-fsst-RFP (RFP, left) orAAV-fsst-Atp23-GFP (Atp23-GFP, right). RFP: Kruskal-Wallis one-way ANOVAon ranks, H=9.373, *P=0.009. WT vs. Rik^(−/−), *P<0.05; WT vs.Rik^(+/−), *P<0.05. Atp23-GFP-Kruskal-Wallis one-way ANOVA on ranks,H=0.09, P=0.956. In FIGS. 4C and 4D, insets show representative tracesof IPSCs (FIG. 4C) and PSPs (FIG. 4D) before (dark traces) and after(light traces) tetanic stimulation. FIGS. 4E and 4F show directinteraction one-chamber test using the same (FIG. 4E) or a different(FIG. 4F) stimulus animal in the 2 trials.

FIGS. 5A-5F show mice carrying a deletion of the 22q11 deletion syndrome(22q11DS) gene 2510002D24Rik are deficient in the social novelty testbut not in sociability. FIG. 5A is a diagram depicting the location ofC22orf39 gene in human chromosome 22 and the mouse orthologue2510002D24Rik in the syntenic region of mouse chromosome 16. The genesdeleted in the Df(16)A^(+/−) mouse model are indicated by the solidblack line at the bottom. In FIG. 5B the 2510002D24Rik mRNA levels arelower in Rik^(+/−) (n=12) and Rik^(−/−) (n=10) mice than in WT mice(n=10). Kruskal-Wallis one-way ANOVA on ranks: H=29.289, *P<0.001. Allpairwise comparisons, *P<0.05. In FIG. 5C, the left panel shows a3-chamber social novelty test. In FIG. 5C, the right panel shows that WTmice (n=14, *P=0.009) but not Rik^(+/−) (n=30, P=0.99) or Rik^(−/−)(n=19, P=0.855) mice preferred the novel mouse over a familiar mouse.Kruskal-Wallis one-way ANOVA on ranks, H=23.084, *P<0.001. FIG. 5D showsthe results of the 3-chamber social preference test. WT (n=11),Rik^(+/−) (n=23), and Rik^(−/−) (n=9) mice explored a novel mouse morecompared to an empty chamber. Two-way RM ANOVA: F(1, 41)=18.05,*P<0.001. No difference was detected among genotypes exploring a novelmouse or an empty chamber (Holm-Sidak pairwise comparison method,P>0.05). FIG. 5E shows that mice carrying a deletion of the 22q11DS gene2510002D24Rik are deficient in the social novelty test but not insociability. (a) Left: 3-chamber social novelty test. Center: WT mice(n=12, *P=0.002) but not Rik+/− (n=21, P=0.441) or Rik−/− (n=15,P=0.777) mice preferred the novel stimulus mouse over a familiarstimulus mouse. Two-way RM ANOVA: F(1,49)=7.486, *P=0.009). Right:Difference score is reduced in 2510002D24Rik-deficient mice in the3-chamber social novelty test. One-way ANOVA: F(2, 46)=3.239, *P=0.048.Pair-wise comparisons, WT vs. Rik+/−, *P=0.032, WT vs. Rik−/−, *P=0.049.In the left panel of FIG. 5F, the 3-chamber social preference test.Center: WT (n=12), Rik+/− (n=23), and Rik−/− (n=9). WT and Rik+/− micesimilarly explored a novel mouse longer than an empty chamber. Two-wayRM ANOVA: F(1,41)=18.049, *P<0.001). In the right panel of FIG. 5F, thedifference scores were similar between WT and 2510002D24Rik-deficientmice in the 3-chamber social preference test. One-way ANOVA: F(2,41)=1.541, P=0.226.

FIGS. 6A-6F shows that the 2510002D24Rik-deficient mice have no deficitsin olfactory investigation, motor function, compulsiveness, or anxiety.FIG. 6A shows that there was no difference among WT (n=8), Rik^(+/−)(n=8), and Rik^(−/−) (n=8) mice in performance of the olfactoryhabituation/dishabituation task. Two-way RM ANOVA: trialF(14,21)=30.392, P<0.001; genotype F(2,21)=0.0368, P=0.964. FIGS. 6B-6Eshow that WT (n=9) Rik^(+/−) (n=9) and Rik^(−/−) (n=10) mice showed nodifference in the open-field exploration task (FIGS. 6B-6C) [time incenter vs. time in corners: two-way RM ANOVA, F(2,25)=0.03, P=0.97. InFIGS. 6D and 6E, the number of bouts center vs corners: two-way RMANOVA, F(2,25)=0.07, P=0.926], in the rotarod task (FIG. 6D) [Left:Kruskal-Wallis one-way ANOVA on ranks, H=2.145, P=0.342. Right one-wayANOVA: F(2,25)=1.281, P=0.295], or grooming time (FIG. 6E) [Left one-wayANOVA, F(2,25)=0.0970, P=0.908. Right: one-way ANOVA, F(2,25)=0.453,P=0.64)]. FIG. 6F shows that no difference was detected in WT (n=9),Rik^(+/−) (n=13), and Rik^(−/−) (n=11) mouse performance in the elevatedplus maze task. Two-way RM ANOVA: location F(2,30)=370.5, P<0.001,genotype F(2,30)=0.942, P=0.401.

FIGS. 7A-7G show that the 2510002D24Rik-deficient mice have no deficitin spatial, contextual, or fear memories. FIGS. 7A-7D show that WT(n=20-22), Rik^(+/−) (n=20-22), and Rik^(−/−) (n=21) mice performedequally well in the spatial memory and visual tasks in the Morris watermaze. The parameters are as follows. Training (FIG. 7A): two-way RMANOVA, genotype F(2,62)=0.521, P=0.596. Visible platform task (FIG. 7B):one-way ANOVA, genotype F(2,58)=2.727, P=0.074. Memory one hour probe(FIG. 7C): two-way RM ANOVA, genotype F(2,58)=0.009, P=0.991. Memory 24hour probe (FIG. 7D): Two-way RM ANOVA, genotype F(2,58)=0.297, P=0.744.FIG. 7E shows that no difference was detected in performance between WT(n=7) and Rik^(−/−) (n=9) mice in the novel object recognition task.Mann-Whitney Rank Sum: U=24, P=0.439. FIGS. 7F and 7G show that WT(n=7), Rik^(+/−) (n=10), and Rik^(−/−) (n=9) mice performed at similarlevels in the contextual and cued versions of the fear-conditioningmemory tasks. The parameters are as follows. Contextual (FIG. 7F):Kruskal-Wallis one-way ANOVA on ranks, genotype H=0.662, P=0.718. Cued(FIG. 7G): two-way RM ANOVA, treatment F(1, 23)=139.59, P<0.001,genotype F(2,23)=0.237, P=0.791.

FIGS. 8A-8F show normal resting membrane potential and excitability in2510002D24Rik-deficient mice. Mean resting membrane potential (FIGS. 8Aand 8D), input resistance (FIGS. 88 and 8E), and rheobase (FIGS. 8C and8F) in CA2 pyramidal neurons (FIGS. 8A-8C) and CA2 interneurons (FIGS.8D-8F). The parameters are as follows. FIG. 8A: Kruskal-Wallis one-wayANOVA on ranks (WT, n=17; Rik^(+/−), n=16; Rik^(−/−), n=21), H=2.691,P=0.26. FIG. 8B: One-way ANOVA (WT, n=17; Rik^(+/−), n=17; Rik^(−/−),n=21), F(2,52)=1.354, P=0.267. FIG. 8C: Kruskal-Wallis one-way ANOVA onranks (WT, n=16; Rik^(+/−), n=17; Rik^(−/−), n=21), H=4.78, P=0.092.FIG. 8D: One-way ANOVA (WT, n=7; Rik^(+/−), n=9; Rik^(−/−), n=8),F(2,21)=0.562, P=0.578. FIG. 8E: One-way ANOVA (WT, n=10; Rik^(+/−),n=10; Rik^(−/−), n=12), F(2,29)=0.55, P=0.583. FIG. 8F: Kruskal-Wallisone-way ANOVA on ranks (WT, n=10; Rik^(+/−), n=10; Rik^(−/−), n=12),H=0.814, P=0.666.

FIGS. 9A-9B show distal inputs from the entorhinal cortex onto CA2 arenot affected in 2510002D24Rik-deficient mice. FIGS. 9A and 9B showaverages of input-output curves of the PSP amplitude in controlcondition (FIG. 9A) and after blocking inhibition with SR95531 andCGP55845 (FIG. 9B) in response to distal stimulation in WT (FIG. 9A: 17neurons, 6 mice; FIG. 9B: 13 neurons, 6 mice), Rik^(+/−) (FIG. 9A: 16neurons, 5 mice; FIG. 9B: 9 neurons, 4 mice), and Rik^(−/−) (FIG. 9A: 20neurons; 5 mice; FIG. 9B: 9 neurons, 4 mice) animals. Two-way RM ANOVA(FIG. 9A): genotype F(2;50)=2.401, P=0.0.101; two-way RM ANOVA (FIG.9B): F(2;28)=0.212, genotype P=0.811). Insets show representativecompound EPSP/IPSP (PSP) traces.

FIGS. 10A-10D show the number of interneurons in the CA2 area of thehippocampus is not altered in 2510002D24Rik-deficient mice. FIG. 10Ashows fluorescent images of the hippocampus stained with antibodiesagainst parvalbumin (left), RGS14, a label of the CA2 area (middle), andmerged image (right) in WT and Rik^(−/−) mice. FIG. 10B is ahigh-magnification of parvalbumin staining in the CA2 area (denoted bylines in a and b and identified by RGS14 staining). FIGS. 10C and 10Dshow mean numbers of PV⁺ interneurons in the CA2 area (FIG. 10C) and theaverage size of the CA2 area in WT and Rik^(−/−) mice (FIG. 10D). Theparameters are as follows. In FIG. 10C, WT, n=4; Rik^(−/−), n=4, t-test,t=0.182, P=0.862. In FIG. 10D, WT, n=2; Rik^(−/−), n=2, Mann-Whitneyrank-sum test, U=2, P=1.

FIG. 11 shows validation of synaptosomal- andmitochondrial-fractionation procedures. The synaptic marker PSD95 andthe mitochondrial marker cytochrome C are expressed in whole (crude)lysate from the hippocampus. Cytochrome C is enriched in themitochondrial fraction, compared to the synaptosomal fractions (note:synaptosomes also contain mitochondria). PSD95 is enriched in thesynaptosomal fractions. Experiments were done in duplicate.

FIGS. 12A, 12B, and 12C show design and validation of the Rik^(HA)knock-in mice. In FIG. 12A, the Rik^(HA) knock-in mouse was produced byinserting the human influenza hemagglutinin (HA) tag upstream of exon 3of the 2510002D24Rik gene by using the CRISPR/Cas9 approach. FIG. 12B isan immunoblot showing presence of HA in Rik^(HA) mice (n=2) but not inWT animals (n=2). FIG. 12C shows fluorescent images of the sagittalsections of the hippocampus in Rik^(HA) mice stained with anti-HA andanti-RGS14 antibodies. The CA2 area of the hippocampus is denoted byRGS14 staining.

FIGS. 13A-13C show activity-dependent changes in ATP and ADP are notsignificantly different in CA2 pyramidal neurons. In FIG. 13A, theexamples (top) and averages (bottom) of PercevalHR fluorescence excitedat 840 nm (ADP) or 940 nm (ATP) before, during, and after 300-pAdepolarization measured through line scans in cell bodies of CA1pyramidal neurons of WT and 2510002D24Rik-deficient mice. FIGS. 13B and13C show mean area under curves (AUC) of PercevalHR fluorescence. Theparameters are as follows. In FIG. 13B, 840 nm-excitation: WT (n=11);Rik^(+/−) (n=8), and Rik^(−/−) (n=11) neurons. Kruskal-Wallis one-wayANOVA on ranks: H=3.809, P=0.149. In FIG. 13C, 940 nm-excitation: WT(n=10); Rik^(+/−) (n=8), and Rik^(−/−) (n=11) neurons. one-way ANOVA:F(2,26)=0.122, P=0.122.

FIG. 14A shows that the 2510002D24Rik protein is localized tomitochondria. Immunostaining for hemagglutinin (HA) and cytochrome C inthe CA2 area from Rik^(HA) sagittal hippocampal sections is shown. FIG.14B shows the number of mitochondria in CA2 inhibitory synapses isunchanged in 2510002D24Rik-deficient mice. Average percent ofmitochondrial-containing excitatory (asymmetric) and inhibitory(symmetric) synapses measured in the CA2 area of the hippocampus of WT(665 and 94 synapses, 2 mice) and Rik^(−/−) (547 and 110 synapses, 2mice) mice. Two-way ANOVA: synapse type, F(1,1)=259.79, P<0.001;genotype, F(1,1)=0.029, P=0.873.

FIGS. 15A and 158 show the design and validation of Atp23 knock-outmice. In FIG. 15A, the Atp23^(+/−) knock-out mouse was produced bydeleting exons 2 and 3 from Atp23 by using the CRISPR/Cas9 approach. InFIG. 15B, average normalized levels of Atp23 mRNA was measured byRT-qPCR in the hippocampus of WT (n=8) and Atp23^(+/−) littermates(n=8). t-test: t=10.229, *P<0.001.

DETAILED DESCRIPTION

The present invention is based on an unexpected discovery that socialmemory deficit can be ameliorated in mice with 22q11DS (particularlymice deficient in 2510002D24Rik-gene product), by ectopic expression ofAtp23 in a CA2 interneuron. Such expression of Atp23 can rescuedeficiencies in interneuron firing, CA3-CA2 synaptic plasticity, andsocial memory in mice deficient in 2510002D24Rik.

Definitions

As used herein, the term “social memory” means the ability of a mammal,including a human, to recognize a subject encountered previously.Recognition may be measured by established tests in the case ofnon-human mammals, or in the case of human mammals, by facialrecognition, behavior, or speech or specific tests measuring differencesin interacting time between novel and familiar animals. The terms“social memory” and “recognition memory” are used interchangeablyherein.

As used herein, the term “schizophrenia” includes a condition generallydescribed as schizophrenia or a condition having symptoms relatedthereto. Schizophrenia can be considered a disease having a spectrum ofmanifestations with various threshold levels. Symptoms of schizophreniamay appear in a range of related disorders including classicalschizophrenia as well as dementia, bipolar disorder, obsessivecompulsive disorder (OCD), panic disorder, phobias, acute stressdisorder, adjustment disorder, agoraphobia without history of panicdisorder, alcohol dependence (alcoholism), amphetamine dependence, briefpsychotic disorder, cannabis dependence, cocaine dependence, cyclothymicdisorder, delirium, delusional disorder, dysthymic disorder, generalizedanxiety disorder, hallucinogen dependence, major depressive disorder,nicotine dependence, opioid dependence, paranoid personality disorder,Parkinson's disease, schizoaffective disorder, schizoid personalitydisorder, schizophreniform disorder, schizotypal personality disorder,sedative dependence, shared psychotic disorder, smoking dependence andsocial phobia.

The terms “vector”, “expression vector”, and “expression construct” areused interchangeably to refer to a composition of matter which can beused to deliver a nucleic acid of interest to the interior of a cell andmediate its expression within the cell. Most commonly used examples ofvectors are autonomously replicating plasmids and viruses (such as,e.g., adenoviral vectors, adeno-associated virus vectors (AAV),adenoviral vectors, retroviral vectors (e.g., lentiviral vectors),Sindbis virus vectors, vaccinia virus vectors, herpes virus vectors,etc.). An expression construct can be replicated in a living cell, or itcan be made synthetically. In one embodiment, an expression vectorcomprises a promoter operably linked to a polynucleotide which promotercontrols the initiation of transcription by RNA polymerase andexpression of the polynucleotide. Typical promoters for mammalian cellexpression include, e.g., SV40 early promoter, CMV immediate earlypromoter (see, e.g., U.S. Pat. Nos. 5,168,062 and 5,385,839), mousemammary tumor virus LTR promoter, adenovirus major late promoter (AdMLP), herpes simplex virus promoter, murine metallothionein genepromoter, and U6 or H1 RNA pol III promoter. Non-limiting examples ofpromoters useful for the methods of the present invention include, e.g.,Synapsin promoter (neuron specific), CamKIIa promoter (specific forexcitatory neurons), CMV promoter, and β-actin promoter. These and otherpromoters can be obtained from commercially available plasmids, usingtechniques well known in the art. See, e.g., Sambrook et al., supra.Enhancer elements may be used in association with promoters to increaseexpression levels of the vectors. Examples include the SV40 early geneenhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, theenhancer/promoter derived from the long terminal repeat (LTR) of theRous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad.Sci. USA (1982b) 79:6777 and elements derived from human CMV, asdescribed in Boshart et al., Cell (1985) 41:521, such as elementsincluded in the CMV intron A sequence.

Typically, transcription terminator/polyadenylation signals will also bepresent in the expression vector. Examples of such sequences include,but are not limited to, those derived from SV40, as described inSambrook et al., supra, as well as a bovine growth hormone terminatorsequence (see, e.g., U.S. Pat. No. 5,122,458). Additionally, 5′-UTRsequences can be placed adjacent to the coding sequence in order toenhance expression of the same. Such sequences include UTRs whichinclude, e.g., an Internal Ribosome Entry Site (IRES) present in theleader sequences of picomaviruses such as the encephalomyocarditis virus(EMCV) UTR (Jang et al. J. Virol. (1989) 63:1651-1660. Other usefulpicornavirus UTR sequences include, e.g., the polio leader sequence,hepatitis A virus leader and the hepatitis C IRES.

In certain embodiments of the invention, the cells containing nucleicacid constructs of the present invention may be identified in vitro orin vivo by including a marker in the expression vector. Such markerswould confer an identifiable change to the cell permitting easyidentification of cells containing the expression vector. Usually theinclusion of a drug selection marker aids in cloning and in theselection of transformants, for example, genes that confer resistance toneomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol areuseful selectable markers. Alternatively, enzymes such as herpes simplexvirus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT)may be employed. Fluorescent markers (e.g., green fluorescent protein(GFP), EGFP, or Dronpa), or immunologic markers can also be employed.Further examples of selectable markers are well known to one of skill inthe art.

In the context of the present invention insofar as it relates to any ofthe disease conditions recited herein, the terms “treat”, “treatment”,and the like mean to relieve or alleviate at least one symptomassociated with such condition, or to slow or reverse the progression ofsuch condition, or to arrest, delay the onset (i.e., the period prior toclinical manifestation of a disease) and/or reduce the risk ofdeveloping or worsening a disease. Within the meaning of the presentinvention, the term “treat” also encompasses preventing and/or reducinga positive symptom associated with schizophrenia or 22q11 DS, such as,e.g., hallucinations, delusions, disorganized thought, or psychosis.

As used herein the term “therapeutically effective” applied to dose oramount refers to that quantity of a compound or pharmaceuticalcomposition that is sufficient to result in a desired activity (e.g.,decrease in positive symptoms associated with schizophrenia and/or22q11DS) upon administration to a subject in need thereof. Note thatwhen a combination of active ingredients is administered, the effectiveamount of the combination may or may not include amounts of eachingredient that would have been effective if administered individually.The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the condition being treated, the particular drug or drugs employed,the mode of administration, and the like. An appropriate “effective”amount in any individual case may be determined by one of ordinary skillin the art using routine experimentation, based upon the informationprovided herein.

The phrase “pharmaceutically acceptable”, as used in connection withcompositions of the invention, refers to molecular entities and otheringredients of such compositions that are physiologically tolerable anddo not typically produce untoward reactions when administered to amammal (e.g., a human). Preferably, as used herein, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in mammals, and moreparticularly in humans.

As used herein, the term “combination” of a composition of the inventionand at least a second pharmaceutically active ingredient means at leasttwo, but any desired combination of compounds can be deliveredsimultaneously or sequentially (e.g., within a 24 hour period). It iscontemplated that when used to treat various diseases, the compositionsand methods of the present invention can be utilized with othertherapeutic methods/agents suitable for the same or similar diseases.Such other therapeutic methods/agents can be co-administered(simultaneously or sequentially) to generate additive or synergisticeffects. Suitable therapeutically effective dosages for each agent maybe lowered due to the additive action or synergy.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the compound is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water or aqueoussolution saline solutions and aqueous dextrose and glycerol solutionsare preferably employed as carriers, particularly for injectablesolutions. Alternatively, the carrier can be a solid dosage formcarrier, including but not limited to one or more of a binder (forcompressed pills), a glidant, an encapsulating agent, a flavorant, and acolorant. Suitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin.

An “individual” or “subject” or “animal”, as used herein, refers tohumans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs,etc.) and experimental animal models of schizophrenia or 22q11 DS. In apreferred embodiment, the subject is a human.

The term “associated with” is used to encompass any correlation,co-occurrence and any cause-and-effect relationship.

The term “about” means within an acceptable error range for theparticular value as determined by one of ordinary skill in the art,which will depend in part on how the value is measured or determined,i.e., the limitations of the measurement system. For example, “about”can mean within an acceptable standard deviation, per the practice inthe art. Alternatively, “about” can mean within an order of magnitude,preferably within 50%, more preferably within 20%, still more preferablywithin 10%, even more preferably within 5%, and most preferably within1% of a given value or range. Alternatively, particularly with respectto biological systems or processes, the term can mean within an order ofmagnitude, preferably within two-fold, of a value. Where particularvalues are described in the application and claims, unless otherwisestated, the term “about” is implicit and in this context means within anacceptable error range for the particular value.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the content clearly dictates otherwise.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition. Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989 (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); Ausubel, F. M. et al.(eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc.,1994. These techniques include site directed mutagenesis as described inKunkel, Proc. Natl. Acad. Sci. USA 82: 488-492 (1985), U.S. Pat. No.5,071,743, Fukuoka et al., Biochem. Biophys. Res. Commun. 263: 357-360(1999); Kim and Maas, BioTech. 28: 196-198 (2000); Parikh andGuengerich, BioTech. 24: 4 28-431 (1998); Ray and Nickoloff, BioTech.13: 342-346 (1992); Wang et al., BioTech. 19: 556-559 (1995); Wang andMalcolm, BioTech. 26: 680-682 (1999); Xu and Gong, BioTech. 26: 639-641(1999), U.S. Pat. Nos. 5,789,166 and 5,932,419, Hogrefe, Strategies 14.3: 74-75 (2001), U.S. Pat. Nos. 5,702,931, 5,780,270, and 6,242,222,Angag and Schutz, Biotech. 30: 486-488 (2001), Wang and Wilkinson,Biotech. 29: 976-978 (2000), Kang et al., Biotech. 20: 44-46 (1996),Ogel and McPherson, Protein Engineer. 5: 467-468 (1992), Kirsch andJoly, Nucl. Acids. Res. 26: 1848-1850 (1998), Rhem and Hancock, J.Bacteriol. 178: 3346-3349 (1996), Boles and Miogsa, Curr. Genet. 28:197-198 (1995), Barrenttino et al., Nuc. Acids. Res. 22: 541-542 (1993),Tessier and Thomas, Meths. Molec. Biol. 57: 229-237, and Pons et al.,Meth. Molec. Biol. 67: 209-218.

The technology illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and use of such terms and expressionsdo not exclude any equivalents of the features shown and described orportions thereof, and various modifications are possible within thescope of the technology claimed.

In one aspect is provided a method for treating or preventing a socialmemory deficit in a subject with a neuropsychiatric disease. One or moreof a therapeutically effective amount of the following is administeredto the subject: (i) a polypeptide encoded by 2510002D24Rik gene or afunctional derivative or fragment thereof, (ii) a vector expressing thepolypeptide encoded by 2510002D24Rik gene or functional derivative orfragment thereof, or (iii) an agent capable of increasing the level oractivity of the polypeptide encoded by 2510002D24Rik gene. In a relatedaspect is provided a method for treating or preventing a social memorydeficit in a subject with a neuropsychiatric disease, the methodcomprising administering to the subject a therapeutically effectiveamount of (i) a protein encoded by 2510002D24Rik gene or a functionalderivative or fragment thereof, or (ii) a vector expressing said proteinencoded by 2510002D24Rik gene or a functional derivative or fragmentthereof.

The human ortholog of 2510002D24Rik gene encodes a protein of unknownfunction and is mapped within the 22q11.2 genomic region (see FIG. 5A).The expression of 2510002D24Rik is strongest in the CA3 and CA2 areas ofthe hippocampus.¹² This application describes how 2510002D24Rik, a geneof previously unknown function, maintains metabolic stability in CA2interneurons that are essential for social memory. Described herein arebehavioral phenotypes present in mice carrying a deletion of the2510002D24Rik gene, with heterozygous and homozygous mice referred tothroughout the application as Rik^(+/−) and Rik^(−/−), respectively).Adult but not juvenile mouse models of 22q11DS encoding a 27-genemicrodeletion that encompasses the 2510002D24Rik gene have a socialmemory deficit associated with neuronal and synaptic dysfunctions in theCA2 area.⁸

In another aspect is provided a method for treating or preventing asocial memory deficit in a subject with a neuropsychiatric disease. Oneor more of a therapeutically effective amount of the following isadministered to the subject: (i) a polypeptide encoded by Atp23 gene ora functional derivative or fragment thereof, or (ii) a vector expressingthe polypeptide encoded by Atp23 gene or functional derivative orfragment thereof, or (iii) an agent capable of increasing the level oractivity of the polypeptide encoded by Atp23 gene. In a related aspectis provided a method for treating or preventing a social memory deficitin a subject with a neuropsychiatric disease, said method comprisingadministering to the subject a therapeutically effective amount of (i) aprotein encoded by Atp23 gene or a functional derivative or fragmentthereof, or (ii) a vector expressing said protein encoded by Atp23 geneor a functional derivative or fragment thereof.

In one embodiment, the present invention provides a method for treatmentand/or prevention of schizophrenia in a subject in need thereof, themethod comprising administering to the subject a therapeuticallyeffective amount of (i) the polypeptide encoded by 2510002D24Rik gene ora functional derivative (including functional fragments) thereof, or(ii) a vector expressing the polypeptide encoded by 2510002D24Rik geneor functional derivative thereof, or (iii) an agent capable ofincreasing the level or activity of the polypeptide encoded by2510002D24Rik gene.

In another embodiment, the present invention provides a method fortreatment and/or prevention of schizophrenia in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of (i) the polypeptide encoded by Atp23gene or a functional derivative (including functional fragments)thereof, or (ii) a vector expressing the polypeptide encoded by Atp23gene or functional derivative thereof, or (iii) an agent capable ofincreasing the level or activity of the polypeptide encoded by Atp23gene.

Atp23 is a mitochondrial intermembrane space protein and is required forthe maturation of the mitochondrially encoded F₀-subunit Atp6 and itsassembly into the F₁F₀-ATP synthase complex.²⁰⁻²² Atp23 is also known asXrcc6bp1, Atp23 metallopeptidase, and ATP synthase assembly factorhomolog. A mouse Atp23 protein is comprised of the sequence:MAGAPGGGELGPAAGEPLLQRPDSGQGSPEPPAHGKPQQGFLSSLFTRDQSCPLMLQKTLDTNPYVKLLLDAMKHSGCAVNRGRHFSCEVCDGNVSGGFDASTSQIVLCENNIRNQAHMGRWTHELIHAFDHCRAHVHWFTNIRHLACSEIRAASLSGDCSLVNELFRLRFGLKQHHQTCVRDRAVLSILAVRNVSREEAQKAVDEVFQTCFNDREPFGRIPHNQTYARYAHRDFQNRDRYYSNI (SEQ ID NO:4). Another mouse Atp23 protein is comprised of the sequence:

(SEQ ID NO: 5) MAGAPGGGELGPAAGEPLLQRPDSGQGSPEPPAHGKPQQGFLSSLFTRDQSCPLMLQKTLDTNPYVKLLLDAMKHSGCAVNRGRHFSCEVCDGNVSGGFDASTSQIVLCENNIRNQAHMGRVVTHELIHAFDHCRAHVHWFTNIRHLACSEIRAASLSGDCSLVNELFRLRFGLKQHHQIETSCVSRPAMNSQSCLGLVS A.The human Atp23 protein is comprised of the sequence: (SEQ ID NO: 2)MAGAPDERRRGPAAGEQLQQQHVSCQVFPERLAQGNPQQGFFSSFFTSNQKCQLRLLKTLETNPYVKLLLDAMKHSGCAVNKDRHFSCEDCNGNVSGGFDASTSQIVLCQNNIHNQAHMNRVVTHELIHAFDHCRAHVDWFTNIRHLACSEVRAANLSGDCSLVNEIFRLHFGLKQHHQTCVRDRATLSILAVRNISKEVAKKAVDEVFESCFNDHEPFGRIPHNKTYARYAHRDFENRDRYYSNI

In another embodiment, the invention provides a method for treatment of22q11 deletion syndrome in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of (i) the polypeptide encoded by 2510002D24Rik gene or afunctional derivative (including functional fragments) thereof, or (ii)a vector expressing the polypeptide encoded by 2510002D24Rik gene orfunctional derivative thereof, or (iii) an agent capable of increasingthe level or activity of the polypeptide encoded by 2510002D24Rik gene.

In another embodiment, the invention provides a method for treatment of22q11 deletion syndrome in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of (i) the polypeptide encoded by Atp23 gene or a functionalderivative (including functional fragments) thereof, or (ii) a vectorexpressing the polypeptide encoded by Atp23 gene or functionalderivative thereof, or (iii) an agent capable of increasing the level oractivity of the polypeptide encoded by Atp23 gene.

In yet another embodiment, the invention provides a method for treatmentof a positive symptom of schizophrenia in a subject in need thereof, themethod comprising administering to the subject a therapeuticallyeffective amount of (i) the polypeptide encoded by 2510002D24Rik gene ora functional derivative (including functional fragments) thereof, or(ii) a vector expressing the polypeptide encoded by 2510002D24Rik geneor functional derivative thereof, or (iii) an agent capable ofincreasing the level or activity of the polypeptide encoded by2510002D24Rik gene.

In yet another embodiment, the invention provides a method for treatmentof a positive symptom of schizophrenia in a subject in need thereof, themethod comprising administering to the subject a therapeuticallyeffective amount of (i) the polypeptide encoded by Atp23 gene or afunctional derivative (including functional fragments) thereof, or (ii)a vector expressing the polypeptide encoded by Atp23 gene or functionalderivative thereof, or (iii) an agent capable of increasing the level oractivity of the polypeptide encoded by Atp23 gene.

In yet another embodiment, the invention provides a method for treatmentof a mental disorder caused by, or aggravated by, a social memorydeficit. The method comprises administering to the subject atherapeutically effective amount of (i) the polypeptide encoded by2510002D24Rik gene or a functional derivative (including functionalfragments) thereof, or (ii) a vector expressing the polypeptide encodedby 2510002D24Rik gene or functional derivative thereof, or (iii) anagent capable of increasing the level or activity of the polypeptideencoded by 2510002D24Rik gene.

In yet another embodiment, the invention provides a method for treatmentof a mental disorder caused by, or aggravated by, a social memorydeficit. The method comprises administering to the subject atherapeutically effective amount of (i) the polypeptide encoded by Atp23gene or a functional derivative (including functional fragments)thereof, or (ii) a vector expressing the polypeptide encoded by Atp23gene or functional derivative thereof, or (iii) an agent capable ofincreasing the level or activity of the polypeptide encoded by Atp23gene.

In various embodiments of the above aspects and embodiments, theadministration results in replenishing Atp23 in CA2 interneurons of thesubject. In some embodiments, the Atp23 in the CA2 interneurons isreplenished to a level of Atp23 found in the CA2 interneurons of ahealthy subject without any deficiency in social memory. In someembodiments, the Atp23 in the CA2 interneurons is replenished to a levelof Atp23 found in the CA2 interneurons of a normal subject. In someembodiments, the Atp23 in the CA2 interneurons is replenished to a levelsufficient to overcome a social recognition deficit. In variousembodiments, the CA2 interneurons are parvalbumin (PV)-positiveinterneurons.

In various embodiments of the above aspects and embodiments, theadministration results in replenishing Atp23 in CA2 area of thehippocampus of the subject. In some embodiments, the Atp23 in the CA2area of the hippocampus is replenished to a level of Atp23 found in theCA2 area of the hippocampus of a healthy subject without any deficiencyin social memory. In some embodiments, the Atp23 in the CA2 area of thehippocampus is replenished to a level of Atp23 found in the CA2 area ofthe hippocampus of a normal subject. In some embodiments, the Atp23 inthe CA2 area of the hippocampus is replenished to a level sufficient toovercome a social recognition deficit.

In various embodiments of the above aspects and embodiments, theneuropsychiatric disease is a schizophrenia spectrum disorder. Invarious embodiments of the above aspects and embodiments, theneuropsychiatric disease is an autism spectrum disorder. In someembodiments, the neuropsychiatric disease is selected fromschizophrenia, 22q11 deletion syndrome, attention-deficit hyperactivitydisorder, generalized anxiety disorder, obsessive-compulsive disorderand autism spectrum disorders (ASD).

In any of the above aspects and embodiments, any viral vector can beused that is capable of accepting the coding sequences for 2510002D24Rikand a polypeptide or protein encoded by Atp23. For example, vectorsderived from adenovirus (AV), adeno-associated virus (AAV), retroviruses(e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus), Sindbisvirus, herpes virus, and the like. The tropism of the viral vectors canbe modified by pseudotyping the vectors with envelope proteins or othersurface antigens from other viruses, or by substituting different viralcapsid proteins, as appropriate. For example, lentiviral vectors of theinvention can be pseudotyped with surface proteins from vesicularstomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectorsof the invention can be made to specifically target certain cells ortissues by engineering the vectors to express certain capsid proteinserotypes. Currently, there are several AAV serotypes available that canbe used for tissue enrichment based on natural tropism toward specificcell types and interaction between different cellular receptors andserotypes. For example, an AAV vector expressing a serotype 2 capsid ona serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene inthe AAV 2/2 vector can be replaced by a serotype 5 capsid gene toproduce an AAV 2/5 vector. Techniques for constructing AAV vectors whichexpress different capsid protein serotypes are within the skill in theart; see, e.g., Rabinowitz J. E. et al. (2002), J Virol 76:791801. Amethod for constructing the recombinant AV vector, and a method fordelivering the vector into target cells, are also described in Xia etal. (2002), Nat. Biotech. 20:1006-1010.

In some embodiments, the vector is selected from the group consisting ofadeno-associated virus (AAV) vectors, adenovirus vectors, retrovirusvectors (e.g., lentivirus vectors), Sindbis virus vectors, vacciniavirus vectors, and herpes virus vectors. In some embodiments, the vectoris an AAV vector. The AAV vector may have a capsid from a serotypeselected from AAV1, AAV2, AAV5, AAV8, or AAV9.

Alternatively, the 2510002D24Rik and the polypeptide encoded by Atp23can be expressed from recombinant circular or linear DNA plasmids usingany suitable promoter, including inducible/regulatable promoters. Insome embodiments, 2510002D24Rik is expressed from a DNA plasmid. In someembodiments, one or more peptides encoded by Atp23 is expressed from aDNA plasmid. In some embodiments, both (i) 2510002D24Rik and (ii) one ormore peptides encoded by Atp23 are expressed from the same DNA plasmid.An IRES sequence may be placed upstream of one of the (i) 2510002D24Rikand (ii) one or more peptides encoded by Atp23.

In various embodiments, the expression of the peptide or functionalderivative or fragment thereof in the vector is controlled by an fsstpromoter. In various embodiments, the expression of the peptide orfunctional derivative or fragment thereof in the vector is controlled bya Synapsin promoter. In various embodiments, the expression of thepeptide or functional derivative or fragment thereof in the vector iscontrolled by a CMV promoter. In various embodiments, the expression ofthe peptide or functional derivative or fragment thereof in the vectoris controlled by a β-actin promoter. In various embodiments, theexpression of the peptide or functional derivative or fragment thereofin the vector is controlled by an hDlx promoter. In various embodiments,the expression of the peptide or functional derivative or fragmentthereof in the vector is controlled by an mDlx promoter. In variousembodiments, the expression of the peptide or functional derivative orfragment thereof in the vector is controlled by a CamKIIa promoter.

The expression of the protein or functional derivative or fragmentthereof in the vector can be controlled by a pan-GABAergic interneuronpromoter. Exemplary pan-GABAergic interneuron promoters include, but arenot limited to, Dlx, the promoter of GABAergic marker GAD67, thepromoter of VGAT (SLC32A1), the promoter of GAD1, and the promoter ofSLC6A1.

Various modes of administration may be undertaken. In some embodiments,the administration is via injection into the CA2 area of the hippocampusof the subject. Alternatively, on some embodiments administration is viaa transcranial surgical injection. Injection may be performed usingvarious syringes. Exemplary syringes that may be used include, but arenot limited to, Hamilton syringes. Ultrathin needles designed forinjecting materials into the brain may also be used. Various micropumpdevices may also be used with syringes and needles. Various otherapparatus for intracerebral drug administration can be used, such asthose described in International Patent Publication Nos. WO2019/088690and WO2009/144287, both of which are incorporated by reference herein intheir entireties.

In some embodiments, administration is systemic. Systemic administrationcan result in delivery to essentially the entire body of an individual.Routes of administration suitable for or treating disorders disclosedherein also include both central and peripheral administration. Centraladministration may result in delivery of a combination to essentiallythe central nervous system of the individual and includes, e.g., nasaladministration, intrathecal administration, epidural administration aswell as a cranial injection or implant. Peripheral administration canresult in delivery of a compound or a combination to essentially anyarea of an individual outside of the central nervous system andencompasses any route of administration other than direct administrationto the spine or brain. The actual route of administration of a compoundor a combination disclosed herein used can be determined by a person ofordinary skill in the art by taking into account factors, including,without limitation, the type of disorder, the cause of the nervoussystem disorder, the severity of the nervous system disorder, theduration of treatment desired, the degree of relief desired, theduration of relief desired, the particular compound used, the rate ofexcretion of the compound used, the pharmacodynamics of the compound orcombination used, the nature of the other compounds to be included inthe combination, the particular route of administration, the particularcharacteristics, history and risk factors of the individual, such as,e.g., age, weight, general health and the like, the response of theindividual to the treatment, or any combination thereof. An effectivedosage amount of a compound disclosed herein can thus readily bedetermined by the person of ordinary skill in the art considering allcriteria and utilizing his best judgment on the individual's behalf.

In various embodiments, the mode of administration is intranasal.

Liquid formulations suitable for injection or for nasal sprays maycomprise physiologically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions and sterile powders forreconstitution into sterile injectable solutions or dispersions.Formulations suitable for nasal administration may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (propylene glycol, polyethyleneglycol (PEG), glycerol,and the like), suitable mixtures thereof, vegetable oils (such as oliveoil) and injectable organic esters such as ethyl oleate. Proper fluiditycan be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersions and by the use of surfactants.

Pharmaceutical formulations suitable for administration by inhalationinclude fine particle dusts or mists, which may be generated by means ofvarious types of metered, dose pressurized aerosols, nebulizers, orinsufflators.

A therapeutically effective amount of the compound may be administeredin a dosing regimen that comprises a single unit dose or multiple unitdoses. For any particular therapeutic peptide or vector, atherapeutically effective amount (and/or an appropriate unit dose withinan effective dosing regimen) may vary, for example, depending on routeof administration, on combination with other pharmaceutical agents.Also, the specific therapeutically effective amount (and/or unit dose)for any particular patient may depend upon a variety of factorsincluding, but not limited to, the disorder being treated and theseverity of the disorder; the activity of the specific pharmaceuticalagent employed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and/or rate of excretion or metabolism of thespecific fusion protein employed; and the duration of the treatment.

In some embodiments, the therapeutically effective dose ranges fromabout 0.005 mg/kg brain weight to 500 mg/kg brain weight, e.g., fromabout 0.005 mg/kg brain weight to 400 mg/kg brain weight, from about0.005 mg/kg brain weight to 300 mg/kg brain weight, from about 0.005mg/kg brain weight to 200 mg/kg brain weight, from about 0.005 mg/kgbrain weight to 100 mg/kg brain weight, from about 0.005 mg/kg brainweight to 90 mg/kg brain weight, from about 0.005 mg/kg brain weight to80 mg/kg brain weight, from about 0.005 mg/kg brain weight to 70 mg/kgbrain weight, from about 0.005 mg/kg brain weight to 60 mg/kg brainweight, from about 0.005 mg/kg brain weight to 50 mg/kg brain weight,from about 0.005 mg/kg brain weight to 40 mg/kg brain weight, from about0.005 mg/kg brain weight to 30 mg/kg brain weight, from about 0.005mg/kg brain weight to 25 mg/kg brain weight, from about 0.005 mg/kgbrain weight to 20 mg/kg brain weight, from about 0.005 mg/kg brainweight to 15 mg/kg brain weight, from about 0.005 mg/kg brain weight to10 mg/kg brain weight.

In some embodiments, the therapeutically effective dose is greater thanabout 0.1 mg/kg brain weight, greater than about 0.5 mg/kg brain weight,greater than about 1.0 mg/kg brain weight, greater than about 3 mg/kgbrain weight, greater than about 5 mg/kg brain weight, greater thanabout 10 mg/kg brain weight, greater than about 15 mg/kg brain weight,greater than about 20 mg/kg brain weight, greater than about 30 mg/kgbrain weight, greater than about 40 mg/kg brain weight, greater thanabout 50 mg/kg brain weight, greater than about 60 mg/kg brain weight,greater than about 70 mg/kg brain weight, greater than about 80 mg/kgbrain weight, greater than about 90 mg/kg brain weight, greater thanabout 100 mg/kg brain weight, greater than about 150 mg/kg brain weight,greater than about 200 mg/kg brain weight, greater than about 250 mg/kgbrain weight, greater than about 300 mg/kg brain weight, greater thanabout 350 mg/kg brain weight, greater than about 400 mg/kg brain weight,greater than about 450 mg/kg brain weight, greater than about 500 mg/kgbrain weight.

In some embodiments, the therapeutically effective dose may also bedefined by mg/kg body weight. As one skilled in the art wouldappreciate, the brain weights and body weights can be correlated.Dekaban A S. “Changes in brain weights during the span of human life:relation of brain weights to body heights and body weights,” Ann Neurol1978; 4:345-56.

Also provided is a container comprising a single dosage form of a stableformulation in various embodiments described herein. In someembodiments, the container is selected from an ampule, a vial, a bottle,a cartridge, a reservoir, a lyo-ject, or a pre-filled syringe. In someembodiments, the container is a prefilled syringe. In some embodiments,the pre-filled syringe is selected from borosilicate glass syringes withbaked silicone coating, borosilicate glass syringes with sprayedsilicone, or plastic resin syringes without silicone. In someembodiments, the stable formulation is present in a volume of less thanabout 50 mL (e.g., less than about 45 mL, 40 mL, 35 mL, 30 mL, 25 mL, 20mL, 15 mL, 10 mL, 5 mL, 4 mL, 3 mL, 2.5 mL, 2.0 mL, 1.5 mL, 1.0 mL, or0.5 mL). In some embodiments, the stable formulation is present in avolume of less than about 3.0 mL.

Also provided are kits or other articles of manufacture which containsthe formulation of the present invention and provides instructions forits reconstitution (if lyophilized) and/or use. Kits or other articlesof manufacture may include a container, an IDDD, a catheter and anyother articles, devices or equipment useful in intrathecaladministration and associated surgery. Suitable containers include, forexample, bottles, vials, syringes (e.g., pre-filled syringes), ampules,cartridges, reservoirs, or lyo-jects. The container may be formed from avariety of materials such as glass or plastic. In some embodiments, acontainer is a pre-filled syringe. Suitable pre-filled syringes include,but are not limited to, borosilicate glass syringes with baked siliconecoating, borosilicate glass syringes with sprayed silicone, or plasticresin syringes without silicone.

The container may hold formulations and further comprise a label on, orassociated with, the container that may indicate directions forreconstitution and/or use. For example, the label may indicate that theformulation is reconstituted to protein concentrations as describedabove. The label may further indicate that the formulation is useful orintended for, for example, intrathecal administration. In someembodiments, a container may contain a single dose of a stableformulation containing a therapeutic agent (e.g., a replacement enzyme).In various embodiments, a single dose of the stable formulation ispresent in a volume of less than about 15 ml, 10 ml, 5.0 ml, 4.0 ml, 3.5ml, 3.0 ml, 2.5 ml, 2.0 ml, 1.5 ml, 1.0 ml, or 0.5 ml. Alternatively, acontainer holding the formulation may be a multi-use vial, which allowsfor repeat administrations (e.g., from 2-6 administrations) of theformulation. Kits or other articles of manufacture may further include asecond container comprising a suitable diluent (e.g., BWFI, saline,buffered saline). Upon mixing of the diluent and the formulation, thefinal protein concentration in the reconstituted formulation willgenerally be at least 1 mg/ml (e.g., at least 5 mg/ml, at least 10mg/ml, at least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml, at least100 mg/ml). Kits or other articles of manufacture may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, IDDDs, catheters,syringes, and package inserts with instructions for use.

In various embodiments, the protein encoded by 2510002D24Rik genecomprises the amino acid sequence which has at least 80% sequenceidentity to SEQ ID NO: 1. In various embodiments, the protein encoded by2510002D24Rik gene comprises the amino acid sequence which has at least83% sequence identity to SEQ ID NO: 1. In various embodiments, theprotein encoded by 2510002D24Rik gene comprises the amino acid sequencewhich has at least 85% sequence identity to SEQ ID NO: 1. In variousembodiments, the protein encoded by 2510002D24Rik gene comprises theamino acid sequence which has at least 87% sequence identity to SEQ IDNO: 1. In various embodiments, the protein encoded by 2510002D24Rik genecomprises the amino acid sequence which has at least 90% sequenceidentity to SEQ ID NO: 1. In various embodiments, the protein encoded by2510002D24Rik gene comprises the amino acid sequence which has at least91% sequence identity to SEQ ID NO: 1. In various embodiments, theprotein encoded by 2510002D24Rik gene comprises the amino acid sequencewhich has at least 92% sequence identity to SEQ ID NO: 1. In variousembodiments, the protein encoded by 2510002D24Rik gene comprises theamino acid sequence which has at least 93% sequence identity to SEQ IDNO: 1. In various embodiments, the protein encoded by 2510002D24Rik genecomprises the amino acid sequence which has at least 94% sequenceidentity to SEQ ID NO: 1. In various embodiments, the protein encoded by2510002D24Rik gene comprises the amino acid sequence which has at least95% sequence identity to SEQ ID NO: 1. In various embodiments, theprotein encoded by 2510002D24Rik gene comprises the amino acid sequencewhich has at least 96% sequence identity to SEQ ID NO: 1. In variousembodiments, the protein encoded by 2510002D24Rik gene comprises theamino acid sequence which has at least 97% sequence identity to SEQ IDNO: 1. In various embodiments, the protein encoded by 2510002D24Rik genecomprises the amino acid sequence which has at least 98% sequenceidentity to SEQ ID NO: 1. In various embodiments, the protein encoded by2510002D24Rik gene comprises the amino acid sequence which has at least99% sequence identity to SEQ ID NO: 1. In some embodiments, the proteinencoded by 2510002D24Rik gene comprises the amino acid sequence SEQ IDNO: 1. In some embodiments, the protein encoded by 2510002D24Rik geneconsists of the amino acid sequence SEQ ID NO: 1.

In various embodiments, the protein encoded by 2510002D24Rik genecomprises the amino acid sequence which has at least 80% sequenceidentity to SEQ ID NO: 3. In various embodiments, the protein encoded by2510002D24Rik gene comprises the amino acid sequence which has at least83% sequence identity to SEQ ID NO: 3. In various embodiments, theprotein encoded by 2510002D24Rik gene comprises the amino acid sequencewhich has at least 85% sequence identity to SEQ ID NO: 3. In variousembodiments, the protein encoded by 2510002D24Rik gene comprises theamino acid sequence which has at least 87% sequence identity to SEQ IDNO: 3. In various embodiments, the protein encoded by 2510002D24Rik genecomprises the amino acid sequence which has at least 90% sequenceidentity to SEQ ID NO: 3. In various embodiments, the protein encoded by2510002D24Rik gene comprises the amino acid sequence which has at least91% sequence identity to SEQ ID NO: 3. In various embodiments, theprotein encoded by 2510002D24Rik gene comprises the amino acid sequencewhich has at least 92% sequence identity to SEQ ID NO: 3. In variousembodiments, the protein encoded by 2510002D24Rik gene comprises theamino acid sequence which has at least 93% sequence identity to SEQ IDNO: 3. In various embodiments, the protein encoded by 2510002D24Rik genecomprises the amino acid sequence which has at least 94% sequenceidentity to SEQ ID NO: 3. In various embodiments, the protein encoded by2510002D24Rik gene comprises the amino acid sequence which has at least95% sequence identity to SEQ ID NO: 3. In various embodiments, theprotein encoded by 2510002D24Rik gene comprises the amino acid sequencewhich has at least 96% sequence identity to SEQ ID NO: 3. In variousembodiments, the protein encoded by 2510002D24Rik gene comprises theamino acid sequence which has at least 97% sequence identity to SEQ IDNO: 3. In various embodiments, the protein encoded by 2510002D24Rik genecomprises the amino acid sequence which has at least 98% sequenceidentity to SEQ ID NO: 3. In various embodiments, the protein encoded by2510002D24Rik gene comprises the amino acid sequence which has at least99% sequence identity to SEQ ID NO: 3. In some embodiments, the proteinencoded by 2510002D24Rik gene comprises the amino acid sequence SEQ IDNO: 3. In some embodiments, the protein encoded by 2510002D24Rik geneconsists of the amino acid sequence SEQ ID NO: 3.

In various embodiments, the protein encoded by Atp23 gene comprises theamino acid sequence which has at least 80% sequence identity to SEQ IDNO: 2. In various embodiments, the protein encoded by Atp23 genecomprises the amino acid sequence which has at least 83% sequenceidentity to SEQ ID NO: 2. In various embodiments, the protein encoded byAtp23 gene comprises the amino acid sequence which has at least 85%sequence identity to SEQ ID NO: 2. In various embodiments, the proteinencoded by Atp23 gene comprises the amino acid sequence which has atleast 87% sequence identity to SEQ ID NO: 2. In various embodiments, theprotein encoded by Atp23 gene comprises the amino acid sequence whichhas at least 90% sequence identity to SEQ ID NO: 2. In variousembodiments, the protein encoded by Atp23 gene comprises the amino acidsequence which has at least 91% sequence identity to SEQ ID NO: 2. Invarious embodiments, the protein encoded by Atp23 gene comprises theamino acid sequence which has at least 92% sequence identity to SEQ IDNO: 2. In various embodiments, the protein encoded by Atp23 genecomprises the amino acid sequence which has at least 93% sequenceidentity to SEQ ID NO: 2. In various embodiments, the protein encoded byAtp23 gene comprises the amino acid sequence which has at least 94%sequence identity to SEQ ID NO: 2. In various embodiments, the proteinencoded by Atp23 gene comprises the amino acid sequence which has atleast 95% sequence identity to SEQ ID NO: 2. In various embodiments, theprotein encoded by Atp23 gene comprises the amino acid sequence whichhas at least 96% sequence identity to SEQ ID NO: 2. In variousembodiments, the protein encoded by Atp23 gene comprises the amino acidsequence which has at least 97% sequence identity to SEQ ID NO: 2. Invarious embodiments, the protein encoded by Atp23 gene comprises theamino acid sequence which has at least 98% sequence identity to SEQ IDNO: 2. In various embodiments, the protein encoded by Atp23 genecomprises the amino acid sequence which has at least 99% sequenceidentity to SEQ ID NO: 2. In some embodiments, the protein encoded byAtp23 gene comprises the amino acid sequence SEQ ID NO: 2. In someembodiments, the protein encoded by Atp23 gene consists of the aminoacid sequence SEQ ID NO: 2.

In various embodiments, the protein encoded by Atp23 gene comprises theamino acid sequence which has at least 80% sequence identity to SEQ IDNO: 4. In various embodiments, the protein encoded by Atp23 genecomprises the amino acid sequence which has at least 83% sequenceidentity to SEQ ID NO: 4. In various embodiments, the protein encoded byAtp23 gene comprises the amino acid sequence which has at least 85%sequence identity to SEQ ID NO: 4. In various embodiments, the proteinencoded by Atp23 gene comprises the amino acid sequence which has atleast 87% sequence identity to SEQ ID NO: 4. In various embodiments, theprotein encoded by Atp23 gene comprises the amino acid sequence whichhas at least 90% sequence identity to SEQ ID NO: 4. In variousembodiments, the protein encoded by Atp23 gene comprises the amino acidsequence which has at least 91% sequence identity to SEQ ID NO: 4. Invarious embodiments, the protein encoded by Atp23 gene comprises theamino acid sequence which has at least 92% sequence identity to SEQ IDNO: 4. In various embodiments, the protein encoded by Atp23 genecomprises the amino acid sequence which has at least 93% sequenceidentity to SEQ ID NO: 4. In various embodiments, the protein encoded byAtp23 gene comprises the amino acid sequence which has at least 94%sequence identity to SEQ ID NO: 4. In various embodiments, the proteinencoded by Atp23 gene comprises the amino acid sequence which has atleast 95% sequence identity to SEQ ID NO: 4. In various embodiments, theprotein encoded by Atp23 gene comprises the amino acid sequence whichhas at least 96% sequence identity to SEQ ID NO: 4. In variousembodiments, the protein encoded by Atp23 gene comprises the amino acidsequence which has at least 97% sequence identity to SEQ ID NO: 4. Invarious embodiments, the protein encoded by Atp23 gene comprises theamino acid sequence which has at least 98% sequence identity to SEQ IDNO: 4. In various embodiments, the protein encoded by Atp23 genecomprises the amino acid sequence which has at least 99% sequenceidentity to SEQ ID NO: 4. In some embodiments, the protein encoded byAtp23 gene comprises the amino acid sequence SEQ ID NO: 4. In someembodiments, the protein encoded by Atp23 gene consists of the aminoacid sequence SEQ ID NO: 4.

In various embodiments, the protein encoded by Atp23 gene comprises theamino acid sequence which has at least 80% sequence identity to SEQ IDNO: 5. In various embodiments, the protein encoded by Atp23 genecomprises the amino acid sequence which has at least 83% sequenceidentity to SEQ ID NO: 5. In various embodiments, the protein encoded byAtp23 gene comprises the amino acid sequence which has at least 85%sequence identity to SEQ ID NO: 5. In various embodiments, the proteinencoded by Atp23 gene comprises the amino acid sequence which has atleast 87% sequence identity to SEQ ID NO: 5. In various embodiments, theprotein encoded by Atp23 gene comprises the amino acid sequence whichhas at least 90% sequence identity to SEQ ID NO: 5. In variousembodiments, the protein encoded by Atp23 gene comprises the amino acidsequence which has at least 91% sequence identity to SEQ ID NO: 5. Invarious embodiments, the protein encoded by Atp23 gene comprises theamino acid sequence which has at least 92% sequence identity to SEQ IDNO: 5. In various embodiments, the protein encoded by Atp23 genecomprises the amino acid sequence which has at least 93% sequenceidentity to SEQ ID NO: 5. In various embodiments, the protein encoded byAtp23 gene comprises the amino acid sequence which has at least 94%sequence identity to SEQ ID NO: 5. In various embodiments, the proteinencoded by Atp23 gene comprises the amino acid sequence which has atleast 95% sequence identity to SEQ ID NO: 5. In various embodiments, theprotein encoded by Atp23 gene comprises the amino acid sequence whichhas at least 96% sequence identity to SEQ ID NO: 5. In variousembodiments, the protein encoded by Atp23 gene comprises the amino acidsequence which has at least 97% sequence identity to SEQ ID NO: 5. Invarious embodiments, the protein encoded by Atp23 gene comprises theamino acid sequence which has at least 98% sequence identity to SEQ IDNO: 5. In various embodiments, the protein encoded by Atp23 genecomprises the amino acid sequence which has at least 99% sequenceidentity to SEQ ID NO: 5. In some embodiments, the protein encoded byAtp23 gene comprises the amino acid sequence SEQ ID NO: 5. In someembodiments, the protein encoded by Atp23 gene consists of the aminoacid sequence SEQ ID NO: 5.

In various embodiments of the above, the subject is human. In certainembodiments, the subject is an adult.

In another aspect is provided a pharmaceutical composition comprising aprotein encoded by 2510002D24Rik gene or a functional derivative orfragment thereof and a pharmaceutically acceptable carrier or excipient.In a related aspect is provided a pharmaceutical composition comprisinga vector encoding a protein encoded by 2510002D24Rik gene or afunctional derivative or fragment thereof and a pharmaceuticallyacceptable carrier or excipient. In yet another aspect is provided apharmaceutical composition comprising a protein encoded by Atp23 gene ora functional derivative or fragment thereof and a pharmaceuticallyacceptable carrier or excipient. In a related aspect is provided apharmaceutical composition comprising a vector encoding a proteinencoded by Atp23 gene or a functional derivative or fragment thereof anda pharmaceutically acceptable carrier or excipient.

Various vectors may be used. Exemplary vectors include, but are notlimited to, adeno-associated virus (AAV) vectors, retrovirus vectors(e.g., lentivirus vectors), adenovirus vectors, Sindbis virus vectors,vaccinia virus vectors, and herpes virus vectors. Lentivirus andadeno-associated virus (AAV) vectors can provide for efficiency andstability when used for gene therapy. The vector may be an AAV vector.Recombinant adeno-associated virus (AAV) vectors are a promisingalternative gene delivery system based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. The AAV vectorsmay be derived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell may be provided by this vector system.(Wagner, et al. (1998) Lancet 351(9117):1702-1703; Keams, et al. (1996)Gene Ther. 9:748-55). Other AAV serotypes, including AAV1, AAV3, AAV4,AAV5, AAV6, AAV8AAV 8.2, AAV9, and AAV rh10 and pseudotyped AAV such asAAV2/8, AAV2/5 and AAV2/6 can also be used. In certain embodiments, theAAV vector has a capsid from a serotype selected from AAV1, AAV2, AAV5,AAV8, and AAV9.

Various retroviral vectors may also be used. In certain embodiments, theretrovirus vector is a lentivirus vector. Recombinant lentiviral vectorsmay advantageously deliver and express peptides in both dividing andnon-dividing mammalian cells.

In various embodiments, in the vector the sequence encoding the proteinencoded by 2510002D24Rik gene or functional derivative or fragmentthereof is operably linked to a promoter selected from the groupconsisting of fsst promoter, hDlx promoter, mDlx promoter, Synapsinpromoter, CMV promoter, β-actin promoter, and CamKIIa promoter. Inrelated embodiments, in the vector the sequence encoding the proteinencoded by Atp23 gene or functional derivative or fragment thereof isoperably linked to a promoter selected from the group consisting of fsstpromoter, hDlx promoter, mDlx promoter, Synapsin promoter, CMV promoter,β-actin promoter, and CamKIIa promoter.

In the vector, the sequence encoding the protein encoded by2510002D24Rik gene or functional derivative or fragment thereof may beoperably linked to a pan-GABAergic interneuron promoter. In the vector,the sequence encoding the protein encoded by Atp23 gene or functionalderivative or fragment thereof may be operably linked to a pan-GABAergicinterneuron promoter.

In the pharmaceutical composition, the protein encoded by 2510002D24Rikgene may comprises the amino acid sequence which has at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In someembodiments, the protein encoded by 2510002D24Rik gene comprises theamino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to SEQ ID NO: 1. In certain embodiments, the protein encoded by2510002D24Rik gene comprises the amino acid sequence SEQ ID NO: 1. Incertain embodiments, the protein encoded by 2510002D24Rik gene consistsof the amino acid sequence SEQ ID NO: 1.

In the pharmaceutical composition, the protein encoded by 2510002D24Rikgene may comprises the amino acid sequence which has at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3. In someembodiments, the protein encoded by 2510002D24Rik gene comprises theamino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to SEQ ID NO: 3. In certain embodiments, the protein encoded by2510002D24Rik gene comprises the amino acid sequence SEQ ID NO: 3. Incertain embodiments, the protein encoded by 2510002D24Rik gene consistsof the amino acid sequence SEQ ID NO: 3.

In the pharmaceutical composition, the protein encoded by Atp23 gene maycomprises the amino acid sequence which has at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity to SEQ ID NO: 2. In some embodiments, theprotein encoded by Atp23 gene comprises the amino acid sequence having80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2. Incertain embodiments, the protein encoded by Atp23 gene comprises theamino acid sequence SEQ ID NO: 2. In certain embodiments, the proteinencoded by Atp23 gene consists of the amino acid sequence SEQ ID NO: 2.

In the pharmaceutical composition, the protein encoded by Atp23 gene maycomprises the amino acid sequence which has at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity to SEQ ID NO: 4. In some embodiments, theprotein encoded by Atp23 gene comprises the amino acid sequence having80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4. Incertain embodiments, the protein encoded by Atp23 gene comprises theamino acid sequence SEQ ID NO: 4. In certain embodiments, the proteinencoded by Atp23 gene consists of the amino acid sequence SEQ ID NO: 4.

In the pharmaceutical composition, the protein encoded by Atp23 gene maycomprises the amino acid sequence which has at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity to SEQ ID NO: 5. In some embodiments, theprotein encoded by Atp23 gene comprises the amino acid sequence having80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5. Incertain embodiments, the protein encoded by Atp23 gene comprises theamino acid sequence SEQ ID NO: 5. In certain embodiments, the proteinencoded by Atp23 gene consists of the amino acid sequence SEQ ID NO: 5.

In various embodiments, the pharmaceutical composition is formulated forinjection into hippocampus. In various embodiments, the pharmaceuticalcomposition is formulated for transcranial surgical injection.

Examples

The present invention is also described and demonstrated by way of thefollowing examples. However, the use of these and other examplesanywhere in the specification is illustrative only and in no way limitsthe scope and meaning of the invention or of any exemplified term.Likewise, the invention is not limited to any particular preferredembodiments described here. Indeed, many modifications and variations ofthe invention may be apparent to those skilled in the art upon readingthis specification, and such variations can be made without departingfrom the invention in spirit or in scope. The invention is therefore tobe limited only by the terms of the appended claims along with the fullscope of equivalents to which those claims are entitled.

Materials and Methods Animals

Mature (16-28 weeks) mice were used for the experiments. All experimentswere carried out using age- and sex-matched mice, except for socialbehavior experiments, where only age-matched male mice were used. Allmouse strains used herein were back-crossed onto the C57BL/6J geneticbackground for at least 5 generations. The care and use of animals werereviewed and approved by the Institutional Animal Care and Use Committeeat St. Jude Children's Research Hospital.

Generation of 2510002D24Rik Knock-Out Mice

The 2510002D24Rik knock-out (KO) (Rik^(+/−) and Rik^(−/−)) mice wereproduced from the embryonic stem (ES) cell clone (12035A-B12,12035A-E11), which was produced by Regeneron Pharmaceuticals, Inc.(Tarrytown, N.Y.) and generated into live mice by KOMP (www.komp.org)and the Mouse Biology Program (www.mousebiology.org) at the Universityof California, Davis. Rik^(+/−) mice were generated using a targetingvector to disrupt the gene downstream of the start codon. Mice were thencrossed with Ella-cre mice (Jackson Labs, Cat No. 003724) expressing agermline-specific cre recombinase to delete the neomycin cassette. Micewere genotyped, as previously described¹², using PCR and the followingprimers: WT Forward Primer: ACC ATG TGA ATC TAC TGC CTG AGG G (SEQ IDNO: 6), WT Reverse Primer: TAT GTG GGT GAA TGC CTG TAG TCC C (SEQ ID NO:7), and Rik Forward Primer: GCT CAC CTA CAC TCT GTG TAT G (SEQ ID NO: 8)and LacZ Reverse Primer: GTG TAG ATG GGC GCA TCG TAA C (SEQ ID NO: 9).Alternatively, the genotyping services of Transnetyx (Cordova, Tenn.)were used. Both Rik^(+/−) and Rik^(−/−) mice reach adulthood withoutgross morphologic abnormalities.

Generation of 2510002D24Rik-HA Knock-In Mice.

The following oligonucleotides were used for cloning sgRNA T7 expressionvector (System Biosciences, Palo Alto, Calif.): 5′-AGG GCC TCC CTC TACCAC AGG AGA-3′ (SEQ ID NO: 10) and 5′-AAA CTC TCC TGT GGT AGA GGG AGG-3′(SEQ ID NO: 11). In vitro transcription was performed using theMEGAshortscript T7 Kit (Thermo Fisher, Waltham, Mass.), and transcribedRNA was purified using MEGAclear RNA Kit (Thermo Fisher). The followingsingle-stranded oligonucleotide was ordered as an Ultramer DNAoligonucleotide from Integrated DNA Technologies (Coralville, Iowa) andused as the DNA donor 5′-TCC AGG CTG CTC AGA AGC ACA CCT TGG TAT GGG CCTTGA GGC AAA GGC CCC CTA CGG ACT GGA ACC TCC CTC TAC CAC AGG AGA AAG ACAAGT ACC CAT ACG ATG TTC CAG ATT ACG CTT GAT AAG CAA CTG CAA CCC TAC CCTTGA CCT AGA CTT TGA CTG GCT CTT ACT TGA CCT GGA ACA CAG AGC AAA GCA TTTCC-3′ (SEQ ID NO: 12).

A mixture of 50 ng/μL sgRNA, 100 ng/μL hCas9 (System Biosciences), and100 ng/μL single-stranded DNA donor was microinjected into the cytoplasmof C57BL6/J zygotes. The injected zygotes were transferred intopseudo-pregnant CD-1 female mice. The F₀ mice were genotyped by PCRusing primers Rik-HA F: 5′-CTC TGT GAG AGC GAG CAG GTT CG-3′ (SEQ ID NO:13) and Rik-HA R: 5′-TCC AGT GGG CAG CAG GCA AT-3′ (SEQ ID NO: 14). PCRproducts were purified, restriction digested with Hpy188III, and thenanalyzed by agarose gel electrophoresis. The sequences of PCR products,with correct sizes of bands, were further confirmed by subcloning andSanger sequencing. F₀ mice with the correct mutation site were crossedwith C57BL/6J mice. The genotype of F₁ mice was confirmed byPCR-restriction-digestion analysis and Sanger sequencing.

Generation of Atp23 Knock-Out Mice

The following oligonucleotides were used for cloning sgRNA1 and sgRNA2T7 expression vectors (System Biosciences):

sgRNA1F- (SEQ ID NO: 15) 5′-AGG GCC AGT AAG AGA GAA GAA CGG-3′ sgRNA1R(SEQ ID NO: 16) 5′-AAA CCC GTT CTT CTC TCT TAC TGG-3′; and sgRNA2F-(SEQ ID NO: 17) 5′-AGG GTC ATA G CG GAT GTG GGA CAG-3′, sgRNA2R-(SEQ ID NO: 18) 5′-AAA OCT GTC CCA CAT CCG CTA TGA-3′.

In vitro transcription was performed using the MEGAshortscript T7 Kitand transcribed RNA was purified using MEGAclear RNA Kit. The followingsingle-stranded oligonucleotide was ordered as an Ultramer DNAoligonucleotide from Integrated DNA Technologies and used as the DNAdonor 5′-CCA TCT TTT TAG CTC CCT TCC TCC CTC CCT CCT TTC CTC TCC CTC CTCAGA GGT GAG AAC TGA ACC TAA GAC CTT GTG CTT ACC AGG CAA GGC CCT ACC ATGGCT GGT GAC GCT GTT CTG CGG TTT CAT GCA CGT TCT CCC GGC TGC TGC TTT GCTGAT GTT CTC TTC TGC CCT CAA GCG TTG AAG TCC CAC AGA CCT TT-3′ (SEQ IDNO: 19).

A mixture of 50 ng/μL sgRNAs, 100 ng/μL hCas9 (System Biosciences) and100 ng/μL single-stranded DNA donors was microinjected into thecytoplasm of C57BL6/J zygotes. The injected zygotes were transferredinto pseudo-pregnant CD-1 female mice. The F₀ mice were genotyped by PCRusing primers:

Atp23 KO-Forward: (SEQ ID NO: 20) 5′-TCCTCCCTCCCTCCTTTCCTCTC-3′;Atp23 KO-Reverse: (SEQ ID NO: 21) 5′-CCTCGGCAGAACACAGAAGAAGAAA-3′; andAtp23 wt-Reverse: (SEQ ID NO: 22) 5′-AATGAGGGTTAGCAAGCAGAAATGTGT-3′.

PCR products were analyzed by agarose gel electrophoresis. The sequencesof PCR products with correct sizes of bands were further confirmed bysubcloning and Sanger sequencing. F₀ mice with the correct mutation sitewere crossed with C57BL6/J mice. The genotype of F₁ mice was confirmedby PCR analysis and Sanger sequencing as described above.

Generation of Mice with Conditional Deletion of 2510002D24Rik

Mice with the floxed allele of 2510002D24Rik (Rik^(+/f) mice) weredesigned by Ingenious Targeting Laboratory (Ronkonkoma, N.Y.). A 14-kbregion used to construct the targeting vector was first subcloned from apositively identified C57BL/6 BAC clone (RP23-127H5) by using ahomologous recombination-based approach. The region was designed suchthat the homologous long arm (LA) extends about 7.2 kb 5′ to the distalLoxP site. The LoxP cassette was inserted 623 bp upstream of exon 1. ALoxP-FRT-flanked Neo cassette was positioned 214 bp downstream of exon3. The targeted region was 4.42 kb, including exons 1-3. The homologousshort arm (SA) extended ˜2.4 kb downstream of the Neo cassette.

The final targeting vector was constructed using conventional cloningand recombineering methods. The targeting vector was confirmed byrestriction analysis after each modification step and by sequencingusing primers designed to read from the Neo-selection cassette into the3′ end of the middle arm of the floxed/targeted region (iNeoN2) and fromNeo to 5′ of the SA (iNeoN3). The single LoxP site was confirmed bysequencing with primer LOX1. Primers T73 and P6 anneal to the vectorsequence and read into the 3′ and 5′ ends, respectively, of the homologyarms.

The targeting vector (10 μg) was linearized and then transfected byelectroporation of HF4 (129/SvEv×C57Bl/6J) (FLP Hybrid) ES cells. Afterselection with G418 antibiotic, surviving clones were expanded for PCRanalysis to identify recombinant ES clones. The Neo cassette in thetargeting vector was removed during ES clone expansion. Screening primerA2 was designed downstream of the SA, outside the 3′ region used togenerate the targeting construct. PCR reactions using A2 with FRTN1Cprimer amplify the 2.69-kb fragment. Five clones were identified aspositive, expanded, and reconfirmed for SA integration. PCR wasperformed on the 5 clones to detect the presence of the distal LoxP siteby using the LOX2 and SDL3 primers. This reaction amplifies a 214-bp WTproduct. The presence of a second PCR product (58 bp larger than the WTproduct) indicates a positive LoxP PCR. Sequencing was performed onpurified PCR DNA by using the LOX2 primer. The presence of the LoxP sitewas confirmed by DNA sequencing.

Secondary confirmation of positive clones identified by PCR wasperformed by Southern blot analysis. To confirm 5′ arm integration, DNAwas digested with SpeI and electrophoretically separated on a 0.8%agarose gel. After transfer to a nylon membrane, the digested DNA washybridized with an MPB3/4 probe targeted against the target region inthe middle arm. DNA from HF4 mouse ES cells was used as a WT control.Positive clones were analyzed by Southern blot analysis for 3′-armintegration. DNA was digested with NcoI and electrophoreticallyseparated on a 0.8% agarose gel. After transfer to a nylon membrane, thedigested DNA was hybridized with probe MPB3/4. Two ES clones wereconfirmed by Southern blotting and were injected into 71 C57BL/6blastocyst stage embryos with 8-12 cells for each clone. Embryos weretransferred to uterus horns of pseudo pregnant CD-1 foster mice matedwith vasectomized B6CBAF1/J studs and developed to term. Of the 19 pupsborn, 14 developed to mostly high male chimeras which were bred to germline transmission.

Genotyping was performed either by PCR using LOX2 and SDL3 primers (WTallele, 214 bp; LoxP allele, 272 bp) or using Transnetyx genotypingservices.

Primers Used for Sequencing:

Primer P6: (SEQ ID NO: 23) 5′-GAG TGC ACC ATA TGG ACA TAT TGT C-3′Primer T73: (SEQ ID NO: 24) 5′-TAA TGC AGG TTA ACC TGG CTT ATC G-3′Primer LOX1: (SEQ ID NO. 25) 5′-CTT GGT CAG GCT GGA AAG AG-3′Primer iNeoN2: (SEQ ID NO. 26) 5′-AGT ATG GCT TTC CTT CCC GAT GG-3′Primer iNeoN3: (SEQ ID NO: 27) 5′-TCT AAG GCC GAG TCT TAT GAG CAG-3′

Primers for PCR Screening:

A2: (SEQ ID NO: 28) 5′-TCC TAG CCA AAT GGA TGG AC-3′ FRTN1C:(SEQ ID NO: 29) 5′-TCG TTC GAA CAT AAC TTC GTA TAG C-3′ LOX2:(SEQ ID NO: 30) 5′-CCA GAT GAT CTA AGT ATA TGT GTT GCA C-3′ SDL3:(SEQ ID NO: 31) 5′-CCT AAC TGG AGA TCA TAA GGT GAG ATG-3′

MPB3/4 Probe Primers:

MPB3: (SEQ ID NO: 32) 5′-CTT CTT TCA CCC TTA GTC ATC CT-3′ MPB4:(SEQ ID NO: 33) 5′-GCA GTT TGG TAC TCA GGA GAG A-3′

Mouse Behavior

Adult (16-26 weeks) male mice were used for all behavioral experiments.The experimenter was blind to genotypes of the mice. For the one-chambersocial behavior tests, the interaction time was scored by an observerblind to genotypes for all experiments, except the one involvingAAV-hDlx-Cre-GFP-injected mice, which was scored by Cleversys SocialScansoftware (Restin, Va.). The three-chamber social behavior test was alsoscored by Cleversys software. All other behavior tests were scored byCleversys Topscan software (Restin, Va.).

One-chamber direct interaction test for social memory. This method wasadapted.¹ Mice were housed in a holding room in 12 hour 12 hourlight-dark cycle. Mice were single-housed for one week beforehabituating to a test arena (i.e., a mouse was allowed to navigate anempty arena for 10 minutes and then was returned to the home cage). Oneday later, the mouse was returned for the sociability test (trial 1).During the sociability test, the mouse was allowed to interact with anovel juvenile (3-4 weeks) male C57Bl/6J mouse for 5 minutes. Theduration of active social interaction between the adult subject mouseand the juvenile stimulus mouse (anogenital and nose-to-nose sniffing,following, and allogrooming initiated by the test subject) was scored byan experimenter blind to the genotypes. After a one hour interval, thetest was run again (trial 2) with either the previously encounteredmouse or a novel juvenile (3-4 weeks) male C57Bl/6J mouse. A decrease insocial interaction time spent in trial 2 with the previously encounteredstimulus mouse compared to that in trial 1 was used as a measure ofsocial (recognition) memory. Time spent interacting with the novelstimulus mouse during trial 2 was used as a measure of sociability. Theduration of interaction was monitored via CleverSys video-acquisitionsoftware. The interaction time during 3-5 minutes of the trial wasrecorded. Mice that interacted for less than 30 seconds duringfamiliarization (first novel mouse) were excluded from analysis.

A three-chamber social sociability and social novelty test was performedas follows. This method was adapted.³⁷ Experiments were performed in athree-chambered apparatus with three evenly spaced compartments(23.6″×15.5″×9.1). During the sociability test, mice were given a choicebetween investigating an empty holding chamber or a holding chamber thatcontains a novel con-specific adult mouse for 10 minutes. A stimulusmouse was placed in the left or right compartments (systematicallyalternated). The subject mouse was placed in the center compartment.After a one hour interval, the subject mouse was tested in a socialnovelty task for 10 minutes. In the social novelty task, the mice weregiven a choice between investigating a new, unfamiliar adult mouse orthe previously investigated (familiar) mouse. The durations ofinteraction between the conspecific or unfamiliar mouse were monitoredvia CleverSys video-acquisition software.

An open-field test was performed as follows. Mice were allowed tonavigate an open field (16″×16″) arena for one hour. The time spent inthe enclosed corners, along the sides, and the center of the arena wereassessed. Locomotor activity was monitored via CleverSysvideo-acquisition software and reported as the number of bouts,duration, and percentage of total time spent in the center and enclosedcorners.

In testing grooming, the number of grooming bouts and duration andpercentage of total time spent grooming were recorded in the open-fieldarena and analyzed using CleverSys software.

A rotarod test was performed as follows. Mice were placed on a rotatingrod accelerating from 4 to 40 rpm in 4 minutes (Manufacturer). The timefor the animal to fall off the rod (latency) and the distance coveredbefore falling were reported.

A Morris water maze test was performed as follows. One hour prior totesting, animals were brought into the testing room and allowed tohabituate. Testing was performed during the animal's inactive phaseunder dim-light conditions. Mice were allowed to navigate in the maze,with swimming patterns recorded using a video camera tracking system(HVS Image, Co., Buckingham, UK) mounted above the pool. Animals learnedto find a hidden, clear platform by using the standard spatial versionof the Morris water maze task for 4 successive days. Each day, animalswere given four 1-minute trials from each starting position with anintertrial latency of one minute. The order of the starting locationswas counterbalanced each day by using a Latin-square design. A spatiallearning (probe) trial was administered one hour after the completion ofspatial training. A spatial memory (probe) trial was administered 48hours after completion of the spatial learning.

During both probe trials, the platform was removed, and the micereceived a single 1-minute trial in which the animal tried to find theescape platform. These trials originated from the starting location thatwas the farthest from the platform's location throughout training. Micealso completed a nonspatial learning task at least seven days aftercompletion of the spatial protocol. In that task, mice were trained tofind a black visible platform for two successive days. During Day 1, theescape platform was located in the same position used during spatialtraining. The next day, the escape platform was moved to a new quadrant.Each day, the mouse was given four 1-minute trials in the same mannerthat occurred during spatial training. To avoid hypothermia, animalswere dried with paper towels and placed in warmed holding cagesimmediately after each round of training and testing trials.

The novel object recognition test was performed as follows. The testingarena consisted of a vinyl, opaque cylinder approximately 40 cm indiameter with walls 40 cm tall. Before recognition memory wasdetermined, the mouse was first habituated to an empty testing arena for3 to 10 minutes. On the day of testing, the mice were habituated to thetesting arena for 3 to 10 minutes, and then returned to their home cagesfor 5 to 10 minutes. Next the mice were placed back in the testing arenafor 3 to 15 minutes with two to four identical objects (T1). Objectsused are custom-fabricated plastic with an overall size of approximately4 cm in height×4 cm in diameter. The mice were then placed back intotheir home cages. One to 48 hours later, each mouse was returned to thetesting arena for 3 to 10 minutes. The mice were then exposed to one tothree identical objects and one novel object (T2). The activity of themice was video recorded and scored using visual-tracking software(CleverSys Inc.) The amount of time the animals explored the novelobject relative to the familiar object was reported.

Fear conditioning testing was performed as follows. Mice wereindividually placed in a conditioning chamber with the room light on andallowed to explore the testing chamber for 2 minutes before a discreteconditioning stimulus (CS) was delivered in the form of a 30 second tone[10 kHz, 75-dB sound pressure level (SPL)]. Within the last 2 seconds ofthe tone, an unconditioned stimulus (US) was delivered in the form of amild foot shock (0.5 mA, 2 s). Mice were allowed to recover for oneminute, and then three more CS-US pairs were delivered. After the lastCS-US pairing, mice remained in the conditioning chamber for one minuteand were then returned to the home cage.

Approximately 24 hours later, mice were placed in a new environment withthe light off and allowed to explore for two minutes, followed byexposure to only the CS tone for 30 seconds. After a recovery period of30 seconds, the tone exposure and recovery period steps were repeatedthree times. The percentage of freezing times during the training periodand during the pre-CS and post-CS periods on the test day were comparedacross groups using Video Freeze software (Med Associates). Forcontextual-fear conditioning, mice were placed in the same environmentas the conditioning day with lights on. The percentage of freezingrelative to the total five minute recording was reported.

An Elevated plus maze test was performed as follows. Mice were testedindividually in the elevated plus maze. The testing apparatus stood 50cm above the floor with two open arms and two closed arms of equaldimensions (50 cm×12 cm) extending away from a square central platform.Plexiglas lateral walls that are perpendicular to the two open armsenclosed the two closed arms. A plexiglass rim (1-cm high) designed toreduce the possibility of falls surrounded the open arms. Mice werereleased facing an open arm and tested for 5 minutes. The amount of timespent in each of the center, open, and closed arms was monitored viaCleverSys video-acquisition software.

Olfactory habituation testing was performed as previously described.³⁸To begin the experiment, mice were individually placed in a clean mousecage and allowed to habituate for one hour. Next, the mouse washabituated to the testing chamber (clear plexiglass chamber measuring 40cm×40 cm×35 cm) for 10 minutes. During the habituation period, a cleancotton swab was presented in 1 of the corners of the testing arena. Forthe test phase, five odors (water, 2 foreign neutral odors (almond,mint) and 2 social scents (male non-con-specifics and femalenon-con-specifics)) were presented three times each for two minutes. Theorder in which these odors were presented is as follows: 3× water, 3×almond, 3× mint, 3× male, 3× female. After each two minute presentation,the mouse was returned to a clean holding cage for one minute. Toprepare test sessions, a 1:100 dilution of the pure almond and mintextracts (McCormick) solutions in H₂O were prepared each day. Cottonapplicators for social scents were made by swapping the bottom of dirtymouse cages containing either male or female mice. The sniffing durationof each mouse at the cotton swab was analyzed with CleverSys TopScanSoftware.

Whole-Cell Electrophysiology

Mouse brains were quickly removed and placed in cold (4° C.) dissectingsolution containing 125 mM choline-CI, 2.5 mM KCl, 0.4 mM CaCl₂, 6 mMMgCl₂, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, and 20 mM glucose (295-300 mOsm),under 95% O₂/5% CO₂. Acute transverse hippocampal slices (400-μm thick)were prepared. After dissection, slices were incubated for 45 minutes inartificial cerebrospinal fluid (aCSF) containing 124 mM NaCl, 2.5 mMKCl, 2 mM CaCl₂, 2 mM MgCl₂, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, and 10 mMglucose (285-295 mOsm), under 95% O₂/5% CO₂ at 32° C. to 34° C. and thentransferred into the submerged recording chamber and superfused (1.5-2mL/min) with aCSF. Electrophysiology and two-photon imaging experimentswere conducted in a perfusion chamber maintained at 32-33° C.Electrophysiological data were acquired using a Multicamp 700Bamplifier, filtered at 2 kHz and digitized at 10/20 kHz using a Digidata1440 digitizer controlled by Clampex acquisition software.

All offline analyses of electrophysiological data was performed inClampfit. Input-output relations of evoked compounds (EPSP/IPSP) in CA2neurons were measured by stimulating the Schaffer collaterals or theentorhinal cortex inputs with a concentric bipolar stimulating electrode(125-μm outer diameter; 12.5-μm inner diameter) connected to an Iso-Flexstimulus isolator with 100-μs pulses. Stimulating and recordingelectrodes were separated by at least 350 to 400 μm. The recordingelectrodes were borosilicate glass capillaries pulled in a Sutter P-1000puller. The recording electrodes had a resistance of approximately 3.5-5mΩ. The CA2 pyramidal neurons were recorded in whole-cell current-clampmode using electrodes filled with 130 mM K gluconate, 10 mM KCl, 0.1 mMEGTA, 2 mM MgCl₂, 5 mM NaCl, 2 mM ATP-Na₂, 0.4 mM GTP-Na, 10 mM HEPES,and 10-25 μM Alexa 594 (pH 7.35, ˜290 mOsm). EPSPs were recorded byholding the cells at −80 mV (accounting for a liquid junction potentialof ˜14 mV). AP firing was recorded directly from fast-spikinginterneurons, which were identified based on their smaller size comparedto pyramidal neurons and electrophysiological properties, such as highermembrane resistance (>150 mΩ) and lower capacitance (<100 pF).

IPSCs were measured in whole-cell voltage-clamp mode in the cells at 0mV (accounting for a liquid junction potential of ˜11 mV) using aCesium-based internal solution of 125 mM CsMeSO₃, 10 HEPES mM, 0.1 mMEGTA, 2 mM CsCl, 5 mM QX314, 5 mM TEA-CI, 10 mM Na₂ creatine phosphate,4 mM MgATP, 0.3 mM NaGTP. Access resistance was monitored using a −5 mVstep after each recording (range, 25-50 mΩ), with cells that displayedunstable access resistance (variations >20%) excluded from the analyses.GABA_(A)R antagonist SR95531 (1 μM) and GABA_(B)R antagonist CGP55845 (1μM) were added to the perfusion aCSF in some experiments, as indicatedin the figures, to block inhibition. D-AP5 (100 μM) and DNQX (10 μM)were added to the perfusion aCSF to measure iLTD. Synaptic plasticity(iLTD and CA2 disinhibition) experiments were performed as previouslydescribed.⁸ High-frequency stimulation (100 pulses at 100 Hz repeatedtwice) was applied after baseline recordings. The magnitude ofplasticity was measured by comparing averaged responses at 30 to 40minutes for iLTD and 35 to 40 minutes for CA2 disinhibition afterinduction, with baseline-averaged responses 10 minutes before induction.

Two-Photon Imaging of PercevalHR

These experiments were performed in acute hippocampal slices (seeabove). Two-photon laser-scanning microscopy was performed using anUltima imaging system (Prairie Technologies, Middleton, Wis.), aTi:sapphire Chameleon Ultra femtosecond-pulsed laser (Coherent Inc.,Santa Clara, Calif.), and 60× (0.9 NA) water-immersion IR objectives(Olympus, Center Valley, Pa.). Changes in fluorescence of PercevalHRwere measured by 12-second line scans spanning a segment of cell bodyand a proximal part of a dendrite. A 300-pA current step was applied for2 seconds to evoke a train of APs. PercevalHR was excited at 940 nm and840 nm for measuring changes in ATP and ADP, respectively. Fluorescencedata from 1-3 scans repeated at 2- to 3-min intervals were averaged.Data were imported into Clampfit for further processing and correctedfor bleaching using the difference between the beginning and the end ofthe 12-s scan. The line-scan traces were filtered at 23 kHz and box-caraveraged. ΔF/F₀ was calculated using a 400-ms baseline (F₀) measuredbefore the onset of the current step.

Subcellular Fractionation of Synaptosomes and Nonsynaptic Mitochondria

Mitochondrial and synaptosomal fractions were prepared using a Percollgradient centrifugation method adapted from previous protocols.^(39,40)Mice were decapitated, and brains were quickly removed and placed incold (4° C.) dissection buffer containing 125 mM choline-CI, 2.5 mM KCl,0.4 mM CaCl₂, 6 mM MgCl₂, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, and 20 mMglucose (295-300 mOsm), under 95% O₂/5% CO₂. Hippocampi from bothhemispheres were isolated in the cold (4° C.) dissection buffer andhomogenized in a Dounce homogenizer with 500 μL mitochondrial isolationbuffer (containing 225 mM sucrose, 75 mM mannitol, 5 mM HEPES, and 1 mMEGTA). Equal volumes of 24% Percoll solution and the isolation bufferwere added to the homogenate to prepare a sample containing 12% Percoll.This sample was layered on a gradient of 26% and 40% Percoll (inisolation buffer) in ultra-clear centrifuge tubes and centrifuged at30,700×g for 10 minutes with slow acceleration and deceleration (nobrakes). The myelin layer accumulated at the top was discarded. The nextlayer containing synaptosomes and the layer between 26% and 40% Percollcontaining mitochondria were aspirated into separate tubes. Theseparated fractions were further diluted with isolation buffer andcentrifuged twice at 16,700×g for 10 minutes to form firm pellets.Protease inhibitor cocktail [cOmplete™ Mini Protease Inhibitor Cocktail,Sigma-Aldrich (St. Louis, Mo.); 1 tablet diluted in 10 mL] was added tothe pellet and stored at −80° C. for Western blot analysis. Forproteomics, fractionated samples were extracted in a urea lysis bufferand stored at −80° C.

Proteomics

Protein digestion and peptide isobaric labeling by tandem mass tags wereperformed. The experiment was performed with a previously publishedprotocol^(41,42) with slight modification. Whole hippocampal tissue orsynaptosomal and mitochondrial fractions, prepared as described above,were extracted in lysis buffer (50 mM HEPES, pH 8.5, 8 M urea, and 0.5%sodium deoxycholate). The protein concentration of the lysates wasdetermined by a Coomassie-stained short gel with bovine serum albumin(BSA) as a standard. Protein (100 μg) for each sample was digested withLysC (Wako Chemicals, Richmond, Va.) at an enzyme-to-substrate ratio of1:100 (w/w) for 2 hours in the presence of 1 mM DTT. Following this, thesamples were diluted to a final 2 M urea concentration with 50 mM HEPES(pH 8.5) and further digested with trypsin (Promega, Madison, Wis.) atan enzyme-to-substrate ratio of 1:50 (w/w) for at least 3 hours.

The peptides were reduced by adding 1 mM DTT for 30 minutes at roomtemperature (RT) followed by alkylation with 10 mM iodoacetamide (IAA)for 30 minutes in the dark at RT. The unreacted IAA was quenched with 30mM DTT for 30 min. Finally, the digestion was terminated and acidifiedby adding trifluoroacetic acid (TFA) to 1%, desalted using C18cartridges (Harvard Apparatus, Cambridge, Mass.), and dried by speedvac. The purified peptides were resuspended in 50 mM HEPES (pH 8.5),labeled with 10-plex TMT reagents (Thermo Fisher) following themanufacturer's recommendation.

Two-dimensional HPLC and mass spectrometry were performed as follows.TMT-labeled samples were mixed equally, desalted, and fractionated on anoffline HPLC (Agilent 1220) using basic pH reverse-phase liquidchromatography (pH 8.0, XBridge C18 column, 4.6 mm×25 cm, 3.5 μmparticle size; Waters, Milford, Mass.). The fractions were dried,resuspended in 5% formic acid, and analyzed by acidic pH reverse-phaseLC-MS/MS analysis. The peptide samples were loaded on a nanoscalecapillary reverse-phase C18 column [new objective, 75 μm ID×˜40 cm, 1.9μm C18 resin (Dr. Maisch GmbH, Ammerbuck-Entringen Germany)] by an HPLCsystem (Waters nanoAcquity), and eluted by a 180-min gradient. Theeluted peptides were ionized by electrospray ionization and detected byan inline Orbitrap Fusion mass spectrometer (Thermo Fisher). The massspectrometer was operated in data-dependent mode with a survey scan inOrbitrap (60,000 resolution, 2×10⁵ AGC target and 50-ms maximal iontime) and MS/MS high-resolution scans (60,000 resolution, 1×10⁵ AGCtarget, 150-ms maximal ion time, 38 HCD normalized collision energy, 1m/z isolation window, and 20-s dynamic exclusion).

Mass spectrometric data analysis was performed as follows. The MS/MS rawfiles were processed by a newly developed tag-based hybrid search engineJUMP, which showed better sensitivity and specificity than commercialpackages (e.g., Proteome Discoverer).³ The data were searched againstthe UniProt mouse database, concatenated with a reversed-decoy databasefor evaluating false-discovery rates. Searches were performed using 10ppm mass tolerance for precursor ions and 15 ppm mass tolerance forfragment ions, fully tryptic restriction with two maximal missedcleavages, three maximal modification sites, and the assignment of a, b,and y ions. TMT tags on lysine residues and N termini (+229.162932 Da)were used for static modifications, with methionine oxidation (+15.99492Da) considered a dynamic modification. MS/MS spectra were filtered bymass accuracy and matching scores to reduce the protein false-discoveryrate to ˜1%. Proteins were quantified by summing reporter ion countsacross all matched peptide-to-spectrum matches using the JUMP softwaresuite.

Immunoprecipitation Assay

Rik^(HA) or WT mouse brains were used for immunoprecipitation assays.Brains were resuspended with RIPA buffer containing protease inhibitorsand sonicated twice at 15% amplitude for 10 seconds in the sonifier(Branson, Danbury, Conn.) on ice. The lysate was then cleared bycentrifugation at 13,000×g for 10 minutes at 4° C. The supernatant wasfirst precleared by incubating with 30 μL protein-G agarose beads (SantaCruz Biotechnology, Dallas, Tex.) for one hour at 4° C. with rotation.Meanwhile, 40 μL of 50% slurry protein-G agarose beads (Santa CruzBiotechnology) were equilibrated with RIPA buffer and incubated with 2μg anti-HA antibody (ab18181, Abcam, Cambridge, Mass.) at RT for onehour. After excess antibody was washed from the beads, precleared brainlysate was added and incubated overnight at 4° C. The beads werecollected by centrifugation and washed three times with RIPA buffer.Bound proteins were mixed with 2× sample buffer containing 10% (v/v)2-mercaptoethanol, and analyzed by performing immunoblotting withspecific antibodies as indicated below.

Western Blotting

The protein sample was fractionated by using SDS-polyacrylamide gelelectrophoresis and transferred onto a PVDF membrane (Thermo Fisher).After incubation with 5% (wt/vol) nonfat dry milk in TBST (10 mM Tris.pH 8.0, 150 mM NaCl, and 0.5% (vol/vol) Tween 20) for 30 minutes,membranes were incubated with antibodies against HA (1:1000 dilution;ab18181, Abcam), ATP23 (1:500 dilution; NBP1-81339, Novus; St. Charles,Mo.), or actin (1:5000 dilution; Sigma-Aldrich) at RT for one hour.Membranes were washed three times for 5 minutes each, and incubated witha 1:3000 dilution of horseradish peroxidase-conjugated anti-mouse oranti-rabbit antibodies (Santa Cruz Biotechnology) at RT for one hour.Blots were washed with TBST three times, and developed by using the ECLsystem (Pierce Biotechnology Inc., Rockford, Ill.).

Quantitative Real-Time PCR

RNA was isolated from the hippocampus by using the mirVana RNA isolationkit (Ambion, Life Technologies, Foster City, Calif.). The iScript cDNASynthesis Kit (Bio-Rad, 170-8891) was used to synthesize cDNA from 1 μgRNA. The following primers were used in the qPCR experiments:

2510002D24Rik: (SEQ ID NO: 34) 5′-GTGTTCCAGGTCAAGTAA-3′ and(SEQ ID NO: 35) 5′-AGAAGGACAAGTGATAAGC-3′, U6: (SEQ ID NO: 36)5′-CGCTTCGGCAGCACATATAC-3′ and (SEQ ID NO: 37)5′-TTCACGAATTTGCGTGTCAT-3′.

RT-qPCR was performed using SYBR green in an Applied Biosystems 7900HTFast Real-time PCR system with the standard protocol. A serial dilutionof cDNA was used to generate a standard curve for each primer set, andthis curve was used to calculate gene concentrations for each sample.All samples were run in duplicate.

Immunohistochemistry and Confocal Imaging

Mice were anesthetized with ethyl carbamate (1.5 g/kg, 25% solution,intraperitoneal) and perfused transcardially with phosphate-bufferedsaline (PBS) for 1-2 minutes and then a fixative (4% paraformaldehyde inPBS). Brains were isolated and stored at 4° C. overnight in the samefixative. Sagittal sections (60-μm thick) were then prepared on a Leicavibratome. Sections with hippocampal regions were chosen forimmunohistochemistry. Sections were permeabilized using 0.3% Triton-XPBS for 30 minutes followed by blocking in 0.3% BSA in PBS. Sectionswere then incubated in primary antibodies for about 24 hours at 4° C.and in secondary antibodies for four to six hours at RT. The secondaryantibodies were washed away using PBS, and then Hoechst nuclear reagent(1:2000) was added for 30 min. Sections were washed using PBS 3-5 timesbetween incubations and mounted on glass slides with 50% glycerol inPBS.

The primary antibodies used were: rabbit polyclonal anti-PV (1:2000,Swant Inc., catalog no. PV25), goat polyclonal anti-PV (1:200, SantaCruzBiotechnology, catalog no. SC7449), mouse anti-reelin (1:1000, R & Dsystems, catalog no. AF3820), rat monoclonal anti-somatostatin (1:200,Millipore, MAB354), rabbit polyclonal anti-ATP23 (XRCC6BP1; 1:200; NovusBiologicals, catalog no. NBP1-81339), mouse monoclonal anti-RGS14(1:250; Neuromab, catalog no. 75-170), chicken anti-GFP (1:500; AbcamInc.). Alexa-conjugated secondary antibodies (1:1000; 488, 555, 647)raised in donkey or goat against primary antibody species were obtainedfrom ThermoFisher. Mounted sections were imaged in a Zeiss LSM780confocal microscope with a pinhole setting of 1 airy disc. Z-stacks oftiled images were stitched using the Zen2.3 software.

For HA immunostaining, the sections were subjected to heat-mediatedantigen retrieval in 0.01 M sodium citrate buffer (pH 6.0) containing0.05% Tween 20 (Sigma) for 20 minutes at 98° C. and cooling for 30minutes at room temperature. Endogenous peroxidase activity wasinhibited by incubating the sections in 3% hydrogen peroxide/water for10 minutes. After blocking with 10% normal goat serum (Vector Labs) inPBS, sections were incubated overnight at 4° C. with rat anti-HA (Roche,Cat #11867423001). HA immunoreactivity was detected using ImmPRESS® HRPanti-rat polymer detection kit [(Vector Labs, Cat #MP-7444), and AlexaFluor™ 488 Tyramide Reagent (Thermo Fisher Scientific, Cat #B40953)].For double labeling, the sections were then incubated with one of thefollowing antibodies: goat anti-PV, rabbit anti-ATP23, mouse anti-RGS14,or anti-CYC. A respective Alexa-594-labeled secondary antibody (ThermoFisher Scientific) was used to reveal the immunocomplexes.

Generation of Plasmids and Viruses

AAV-hsyn-PercevalHR was generated as follows. To generate AAVsexpressing syn-Perceval, PCR was used to amplify the human synapsinpromoter (hSyn) from pAAV-6P-SEWB. The pAAV-GFP (Addgene, plasmid 32395)was cut with SnaB1 and Sac1 to replace CMV with hSyn (pAAV-hSyn-GFP).The PCR product of PercevalHR from plasmid GW1-PercevalHR (Addgeneplasmid 49082) was cut with Sal1 and EcoR1, and then ligated intopAAV-hsyn-GFP.

pAAV-fsst-GFP-T2A-Atp23 and pAAV-hdIx-Cre-T2A-GFP was generated asfollows. Coding sequences of the Atp23(NM_001159559.1) were amplifiedwith the following primers: Atp23 F HindIII:5-TAAAGCTTATGGCAGGAGCTCCGG-3′ (SEQ ID NO: 38) and ATP23 R HindIII:5′-TAAAGCTTTCATATGTTGGAGTAGTAG-3′ (SEQ ID NO: 39) from cDNA generatedfrom reverse-transcribed mouse whole-brain RNA using the SuperscriptFirst-Strand Synthesis RT-PCR kit (Invitrogen, Carlsbad, Calif.),subcloned into pAAV-CamKII-GFP-2A vector plasmid (from the St. JudeVector Core) after cutting with HindIII. Then GFP-T2A-Atp23 was replacedwith RFP of pAAV-fSST-RFP plasmid (Addgene, plasmid 22913) to create thepAAV-fSST-GFP-T2A-Atp23 plasmid. The pAAV-fsst-RFP was purchased fromAddgene (Watertown, Mass.; plasmid 22913).

pAAV-hDlx-Cre-T2A-eGFP was generated as follows. The Cre-T2A-eGFPfragment was PCR-amplified from pAAV-CMV-Cre-T2A-eGFP by using 2 PCRprimers, Cre F SaII: 5′-ATGTCGACCACCATGTCCAATTTACTGACC-3′ (SEQ ID NO:40) and eGFP R AscI 5′-ATGGCGCGCCTTACTTGTAAAGCTCGTC-3′ (SEQ ID NO: 41).The fragment was then digested with SaII and AscI, and then insertedbetween the SaII and AscI sites of pAAV-hDlx-Flex-GFP-Fishell_6(Addgene, plasmid 83895).

Stereotaxic Injection of AAV Constructs

Mice were anaesthetized using isoflurane (2% for induction and 1.5% formaintenance) in 100% oxygen, with their heads restrained on astereotaxic apparatus. An approximately 1-cm midline incision was madecentered about 0.25 cm behind bregma. Viruses were injected into 2locations within the CA2 region, in one or both hemispheres. Thestereotaxic coordinates for the two injections in relation to the bregmawere as follows: (1) −1.6 mm anteroposterior, 1.6 mm lateral, and 1.7 mmdeep; (2) 2 mm anteroposterior, 2.6 mm lateral, and 1.8 mm deep. Forbehavioral experiments, injections were restricted to the anteriorlocation in both hemispheres. Craniotomy holes were drilled at theselocations, and 300 nL of AAVs was slowly (30 nL/min) injected via a 33Gcannula. After each injection, the cannula was left in place for 2-3minutes before being retracted. Following injections, the skin wassutured, and the mice were returned to the holding cages after recoveryfrom anesthesia. Imaging and electrophysiology experiments wereperformed 4-7 weeks after AAV injections. During each experiment, carewas taken to limit the differences in post-injection duration to amaximum of 5 days across experimental groups to avoid substantialdifferences in the levels of AAV expression.

Electron Microscopy

Mice were anesthetized with ethyl carbamate (1.5 g/kg, 25% solution,intraperitoneal) and then perfused transcardially with PBS for 1-2minutes and then with a fixative (2.5% glutaraldehyde and 2%paraformaldehyde in 0.2 M sodium cacodylate). Brains were isolated,stored at 4° C. overnight in the same fixative, and sagittal sections(100-μm thick) were prepared on a Leica vibratome. Smaller regions (˜500μm×500 μm) containing the stratum radiatum of the CA2 hippocampal regionwere processed for 3D transmission electron microscopy. The samples werestained using a modified heavy metal-staining method, processed througha graded series of alcohol and propylene oxide, and then embedded inEpon hard resin.⁴⁴ Sections (0.5-μm thick) were cut to determine thecorrect area and then coated with iridium in a Denton Desk II sputtercoater. The 3D electron microscopy images were collected on a HeliosNanolab 660 Dualbeam system. From the 3D stacks of electron micrographs(5×5×10 nm³ voxel size, 250-260 sections of 10-nm thickness andapproximately 30×20 μm² area), synapses were identified based on thepresence of postsynaptic densities and presynaptic vesicles. The 3Dstacks were then analyzed using the cell-counter plugin in ImageJ.Representative movies of asymmetric and symmetric synapses containingmitochondria were generated using Amira 6.0 software.

Other Drugs and Chemicals

SR95531, CGP55845, D-AP5, and DNQX were obtained from Tocris Bioscience(Minneapolis, Minn.). Stock solutions of these drugs were prepared inmanufacturer-recommended solvents and stored at −20° C.

Quantification and Statistical Analyses

Data are presented as mean±SEM in dot or bar graphs and median with25^(th) to 75th percentile box plots and error bars denoting 10th and90^(th) percentiles. Statistical analyses were performed using Sigmaplot12.5 software. Statistical analyses were carried out using data fromsingle cells for electrophysiological experiments and mice forbehavioral and molecular experiments. Parametric or nonparametric testswere chosen based on the normality and variance of data distribution.Independent or paired t-tests, Mann-Whitney Rank-Sum test, One-WayANOVA/Kruskal Wallis one-way ANOVA on ranks followed by amultiple-comparison procedure (Dunn's method), two-way ANOVA followed byHolm-Sidak multiple-comparison procedure, one/two-way RM ANOVA were thestatistical tests used. Differences with P<0.05 were consideredsignificant.

Results

Silencing or lesioning the CA2 area of the murine hippocampus is knownto cause a specific deficit in social-recognition memory, with no changein sociability or spatial and contextual memories typically associatedwith the hippocampus.^(1,5) Described herein are similar behavioralphenotypes present in mice carrying a deletion of the 2510002D24Rik gene(Rik^(+/−) and Rik^(−/−) mice), the expression of which is strongest inthe CA3 and CA2 areas of the hippocampus.¹² Adult (>16 weeks) mice wereused in the experiments. As described below, the expression of2510002D24Rik was reduced about 50% in Rik^(+/−) mice, and was notdetected in Rik^(−/−) mice (see FIG. 5B). Both mutants were deficient insocial memory measured in the one-chamber direct-interaction and3-chamber social novelty tests.^(1,15)

In the direct-interaction test, a subject mouse was exposed to anunfamiliar mouse in the first trial (trial 1). After a one hourintertrial interval, the subject mouse was either re-exposed to the samemouse (social recognition/memory test) or another unfamiliar mouse(sociability test) in trial 2. WT, Rik^(+/−), and Rik^(−/−) mice weretested as subject mice. The investigation time for each subject mousewas tested. The data is shown in FIG. 1A for the same mouse, and in FIG.1B for the unfamiliar mouse.

Social memory, measured as the decreased time a subject mouse spendsexploring a previously encountered mouse, was reduced in both mutantscompared to wild-type (WT) controls, as shown in FIG. 1A. In contrast,mutant mice were not significantly different from WT littermates insociability, as shown in FIG. 1B. Rik^(+/−), Rik^(−/−), and WT subjectmice showed similar, unchanging exploration durations during trials 1and 2 when different novel mice were encountered in the 2 trials.

Social memory deficit was confirmed in the social novelty test in bothmutants. In this test, social recognition was measured by the increasedtime that a subject spent interacting with a novel unrelated mouse,compared with that spent interacting with a familiar littermate (FIG.5C, left). WT mice demonstrated a significant preference for thecompartment containing the novel animal, whereas Rik^(−/−) and Rik^(−/−)mice did not (FIG. 5C, right). This deficit was not due to a decrease insociability because the subject mice of all three genotypes showed asimilar preference for a chamber containing a novel mouse versus anempty chamber (FIG. 5D).

Rik^(+/−) and Rik^(−/−) mice behaved like WT controls in detecting andrecognizing nonsocial and social odors (data shown in FIG. 6A), which iscrucial for social interaction.¹⁶ Furthermore, both mutants performed atthe WT level in the open-field, rotarod, grooming, and elevatedplus-maze tests, as shown in FIGS. 6B-6F. Rik^(+/−) and Rik^(−/−) micealso performed comparably to WT mice in hippocampus-dependent memorytasks assayed by the Morris water maze test (data shown in FIGS. 7A-7D),the novel object-recognition test (data shown in FIG. 7E), and fearmemory tasks assayed by contextual and cued-fear conditioning tests(data shown in FIGS. 7F and 7G).

Social memory deficits in a 22q11DS mouse model (Df(16)A^(+/−) mice)¹⁷were attributed to reduced inhibitory control of the excitatory drive inCA2 pyramidal neurons via reduction of numbers and function ofparvalbumin (PV)-positive interneurons in the CA2 area.$ Whole-cellcurrent-clamp experiments in acute hippocampal slices revealed that theintrinsic electrical properties such as resting membrane potential,input resistance, and rheobase of CA2 pyramidal or fast-spikinginterneurons did not differ between 2510002D24Rik-deficient mice and WTcontrols, as shown in FIGS. 8A-8F. However, the inhibitory control atCA3-CA2 excitatory synapses was reduced in 2510002D24Rik-deficient mice.Postsynaptic potentials (PSPs) were recorded in CA2 pyramidal neurons,with or without GABA-receptor antagonists (SR95531 and CGP55845), inresponse to stimulation of Schaffer collaterals.

As seen in Df(16)A^(+/−) mice⁸, the PSP peak amplitudes weresignificantly larger in both mutants than WT controls at basalcondition, as shown in FIG. 1C. This difference disappeared in thepresence of GABA-receptor antagonists (see FIG. 1D). Similar to what wasseen in Df(16)A^(+/−) mice⁸, this increase in PSPs was specific toproximal CA3 inputs and absent in distal inputs from the entorhinalcortex, as shown in FIGS. 9A and 9B. The data of FIG. 1E shows thatinhibitory PSPs (IPSPs) were also significantly smaller in2510002D24Rik-deficient mice than in WT controls. Together, theseresults suggest that 2510002D24Rik-deficient mice have defectiveinhibitory control of the excitatory drive in CA2 pyramidal neurons.

Unlike Df(16)A^(+/−) mice, 2510002D24Rik-deficient mice have a normalnumber of PV⁺ interneurons in the CA2 area, as shown in FIGS. 10A-10D.However, the CA2 fast-spiking interneurons of 2510002D24Rik-deficientmice fired fewer action potentials (APs) in response to an injection ofa depolarizing current step, compared to WT controls. The firing rate ofthese interneurons was measured by injecting a step current incurrent-clamp mode. A depolarizing current (150 pA, 1 s) evoked fewerAPs in 2510002D24Rik-deficient interneurons than in WT interneurons, asshown in FIG. 1F. This suggests that a decrease in inhibitory control ofthe excitatory drive in CA2 pyramidal neurons is mediated by theinability of CA2 interneurons to sustain firing of APs.

Inhibitory control is essential for disinhibitory plasticity at CA3-CA2excitatory synapses. Typically, CA2 pyramidal neurons cannot undergolong-term potentiation¹⁸, but long-term depression at CA3-CA2 inhibitorysynapses (CA3-CA2 iLTD) allows for an increase in the net excitatorydrive from Schaffer collaterals (CA3 neurons) onto CA2 pyramidal neurons(long-term disinhibitory plasticity).¹⁹ In Df(16)A^(+/−) mice, bothCA3-CA2 iLTD and disinhibitory plasticity at CA3-CA2 excitatory synapseswere reduced.⁸ Similarly, both CA3-CA2 iLTD and long-term disinhibitoryplasticity were impaired in 2510002D24Rik-deficient mice (FIGS. 1C-1H).CA3-CA2 iLTD induced by stimulating the Schaffer collaterals withhigh-frequency trains (2×100 Hz) was impaired in Rik^(+/−) and Rik^(−/−)mutants compared to WT controls (FIG. 1G). Long-term disinhibitoryplasticity in CA2 pyramidal neurons evoked by the same stimulationprotocol was also significantly lower in Rik^(+/−) and Rik^(−/−) micethan in WT controls (FIG. 1H).

Unbiased proteomics analysis of the whole hippocampus of Rik^(−/−) miceand WT littermates revealed that the 3 most downregulated proteins inthe mutants were a polypeptide encoded by 2510002D24Rik and 2 peptidesencoded by Atp23 (also known as Xrcc6bp1, Atp23 metallopeptidase, andATP synthase assembly factor homolog). The data are shown in FIG. 2A andTable 1.

TABLE 1 Proteins in the whole hippocampus of 16-week-old Rik^(−/−) miceare altered compared to those in WT littermates. Gene Protein Accession# Log2FC P-value Atp23 sp|Q9CWQ3|ATP23_MOUSE −1.881174 0.0001112510002D24Rik tr|A0A087WSJ3|A0A087WSJ3_MOUSE −1.443354 0.000374 NAsp|P03987|IGHG3_MOUSE −0.958906 0.017582 Tmem27 sp|Q9ESG4|TMM27_MOUSE−0.682104 0.00066 Sult1c2 sp|Q9D939|ST1C2_MOUSE −0.631535 0.013425 Krt8sp|P11679|K2C8_MOUSE −0.454765 0.001507 Ide tr|F6RPJ9|F6RPJ9_MOUSE−0.432014 0.004642 Ide sp|Q9JHR7|IDE_MOUSE −0.430591 0.004747 Lgals3sp|P16110|LEG3_MOUSE −0.429093 0.001087 Kcnj13 sp|P86046|KCJ13_MOUSE−0.422139 0.000994 Agt sp|P11859|ANGT_MOUSE −0.393425 0.002566 Grk5sp|Q8VEB1|GRK5_MOUSE −0.379441 0.019111 Adh1 sp|P00329|ADH1_MOUSE−0.378093 0.002681 Tmem72 sp|Q8C3K5|TMM72_MOUSE −0.37074 0.001613 Itgb4sp|A2A863|ITB4_MOUSE −0.367451 0.010811 Gch1 sp|Q05915|GCH1_MOUSE−0.331395 0.004176 Junb sp|P09450|JUNB_MOUSE −0.321878 0.003634 Arhgdibsp|Q61599|GDIR2_MOUSE −0.309459 0.018167 Myo7a sp|P97479|MYO7A_MOUSE−0.30626 0.016237 Nudt3 sp|Q9JI46|NUDT3_MOUSE −0.271961 0.005056 Cgnsp|P59242|CING_MOUSE −0.255429 0.007692 Galm sp|Q8K157|GALM_MOUSE−0.251655 0.006792 Vat1l sp|Q80TB8|VAT1L_MOUSE −0.247896 0.011458Spata18 sp|Q0P557|MIEAP_MOUSE −0.246716 0.018788 Slc25a1sp|Q8JZU2|TXTP_MOUSE −0.246431 0.009027 Gm11992 sp|Q5SS90|CG057_MOUSE−0.243045 0.010784 Ptpn14 sp|Q62130|PTN14_MOUSE −0.235317 0.014325Pcdhb16 tr|Q91Y03|Q91Y03_MOUSE −0.231047 0.016708 Atg5sp|Q99J83|ATG5_MOUSE −0.229725 0.012045 Sema3b sp|Q62177|SEM3B_MOUSE−0.224553 0.012053 Pus7 tr|Q91VU7|Q91VU7_MOUSE −0.221852 0.01531 Hfesp|P70387|HFE_MOUSE −0.216421 0.019498 Shq1 sp|Q7TMX5|SHQ1_MOUSE−0.214661 0.012722 Klhdc4 tr|G3X961|G3X961_MOUSE −0.208328 0.014303 Crb2sp|Q80YA8|CRUM2_MOUSE −0.2068 0.019946 Wscd1 sp|Q80XH4|WSCD1_MOUSE−0.206734 0.01736 Chuk sp|Q60680|IKKA_MOUSE −0.205388 0.01878 Thrspsp|Q62264|THRSP_MOUSE −0.203686 0.015395 Oprm1 sp|P42866|OPRM_MOUSE−0.203028 0.014209 Ttc25 sp|Q9D4B2|TTC25_MOUSE −0.202911 0.013687Pcdhb11 tr|Q91UZ8|Q91UZ8_MOUSE −0.202469 0.013802 Sars2sp|Q9JJL8|SYSM_MOUSE −0.190878 0.018022 Tspyl4 sp|Q8VD63|TSYL4_MOUSE−0.182806 0.018933 Kpna2 sp|P52293|IMA1_MOUSE 0.19299 0.016252 Pak6sp|Q3ULB5|PAK6_MOUSE 0.19591 0.019609 P4htm sp|Q8BG58|P4HTM_MOUSE0.19597 0.015938 Itm2a sp|Q61500|ITM2A_MOUSE 0.20647 0.014504 Gm16494tr|E9Q2Z4|E9Q2Z4_MOUSE 0.21461 0.012472 Sts sp|P50427|STS_MOUSE 0.215930.014673 Plekhd1 sp|B2RPU2|PLHD1_MOUSE 0.21844 0.017192 Med24sp|Q99K74|MED24_MOUSE 0.22488 0.016817 Grpel2 sp|O88396|GRPE2_MOUSE0.24109 0.01104 Rnf146 sp|Q9CZW6|RN146_MOUSE 0.24268 0.011273 Atxn2tr|F6V8M6|F6V8M6_MOUSE 0.24786 0.007166 Yjefn3 sp|F6W8I0|YJEN3_MOUSE0.25565 0.00703 H1f0 sp|P10922|H10_MOUSE 0.25935 0.011419 Zfp37sp|P17141|ZFP37_MOUSE 0.25956 0.018573 Zscan21 sp|Q07231|ZSC21_MOUSE0.26003 0.011638 Itga1 sp|Q3V3R4|ITA1_MOUSE 0.29782 0.01023 Itga3sp|Q62470|ITA3_MOUSE 0.2981 0.00918 Fam219b sp|Q14DQ1|F219B_MOUSE0.32615 0.003074 Sypl2 sp|O89104|SYPL2_MOUSE 0.32904 0.018114 Chic1sp|Q8CBW7|CHIC1_MOUSE 0.35784 0.018756 Zkscan16 tr|A2ALW2|A2ALW2_MOUSE0.42337 0.005996 Cox17 sp|P56394|COX17_MOUSE 0.45677 0.001197 Neu3sp|Q9JMH7|NEUR3_MOUSE 0.46695 0.002894 Gm4975 tr|J3QMK1|J3QMK1_MOUSE0.6986 0.01093 Efcab7 sp|Q8VDY4|EFCB7_MOUSE 1.16573 0.002172 Tmem126bsp|Q9D1R1|T126B_MOUSE 1.19056 3.93E−05 Nnt sp|Q61941|NNTM_MOUSE 2.354699.43E−06

The same three proteins were also the most downregulated in validatedsynaptosomal fractions (see FIG. 11) isolated from the hippocampi ofRik^(−/−) and WT mice, as shown in FIG. 2B and Table 2. Western-blotanalysis confirmed that Atp23 is substantially reduced in Rik^(−/−)mice, as shown in FIG. 2C.

TABLE 2 Altered proteins in synaptosomes extracted from the wholehippocampus of 16-week old Rik^(−/−) mice compared to WT littermates.Gene Protein Accession # Log₂FC p-value Atp23 sp|Q9CWQ3|ATP23_MOUSE−2.49129 4.93697E−06 2510002D24Rik tr|A0A087WSJ3|A0A087WSJ3_MOUSE−2.135615 6.13385E−06 Klhdc10 sp|Q6PAR0|KLD10_MOUSE −0.7861070.007443791 Myo15a sp|Q9QZZ4|MYO15_MOUSE −0.637775 0.011584436 Ces1sp|Q8VCC2|EST1_MOUSE −0.464792 0.005176465 Psrc1 sp|Q9D0P7|PSRC1_MOUSE−0.452784 0.006437234 Caln1 sp|Q9JJG7|CABP8_MOUSE −0.429955 0.017719806Mxra7 sp|Q9CZH7|MXRA7_MOUSE −0.402925 0.018451731 Dspsp|E9Q557|DESP_MOUSE −0.402163 0.013154723 Nod2 sp|Q8K3Z0|NOD2_MOUSE−0.401952 0.012247843 Cfap58 tr|B2RW38|B2RW38_MOUSE −0.3905680.010277119 Pigu sp|Q8K358|PIGU_MOUSE −0.378273 0.019258143 Cdc42ep2sp|Q8JZX9|BORG1_MOUSE −0.377048 0.007201631 Senp5 sp|Q6NXL6|SENP5_MOUSE−0.351623 0.013507216 Cd44 sp|P15379-10|CD44_MOUSE −0.346483 0.016043442Gnptab sp|Q69ZN6|GNPTA_MOUSE −0.343222 0.001065776 Klhl2sp|Q8JZP3|KLHL2_MOUSE −0.333578 0.002589927 Cystm1 sp|Q8K353|CYTM1_MOUSE−0.33277 0.005635468 Lman2l sp|P59481|LMA2L_MOUSE −0.330438 0.00653487Casd1 sp|Q7TN73|CASD1_MOUSE −0.307547 0.013714455 Rhousp|Q9EQT3|RHOU_MOUSE −0.300097 0.016850365 Ubxn4tr|A0A087WSK5|A0A087WSK5_MOUSE −0.29182 0.003650985 Btbd3sp|P58545|BTBD3_MOUSE −0.289489 0.003578013 Ypel3 sp|P61237|YPEL3_MOUSE−0.286072 0.009207956 Gc sp|P21614|VTDB_MOUSE −0.275213 0.019945805Hspa13 sp|Q8BM72|HSP13_MOUSE −0.274189 0.009343557 Arhgap42sp|B2RQE8|RHG42_MOUSE −0.262866 0.00548731 Samd8 sp|Q9DA37|SAMD8_MOUSE−0.256451 0.01427959 Slco3a1 sp|Q8R3L5|SO3A1_MOUSE −0.256172 0.014865082Kcnk4 sp|O88454|KCNK4_MOUSE −0.24736 0.015664998 Tor2asp|Q8R1J9|TOR2A_MOUSE −0.244306 0.018760002 Sumf2 sp|Q8BPG6|SUMF2_MOUSE−0.242812 0.016631124 D17h6s53e sp|Q9Z1R4|CF047_MOUSE −0.2400270.00661586 Gigyf2 sp|Q6Y7W8|PERQ2_MOUSE −0.239663 0.012603122 Cerksp|Q8K4Q7|CERK1_MOUSE −0.231491 0.012358048 Ephx4 sp|Q6IE26|EPHX4_MOUSE−0.226445 0.012088793 Tyk2 sp|Q9R117|TYK2_MOUSE −0.225768 0.007738916Dhrs13 sp|Q5SS80|DHR13_MOUSE −0.22231 0.013625179 Nfx1sp|B1AY10|NFX1_MOUSE −0.204414 0.018345683 Dennd2a sp|Q8C4S8|DEN2A_MOUSE−0.201213 0.018636601 Qsox2 sp|Q3TMX7|QSOX2_MOUSE −0.200757 0.018927503Rnf181 sp|Q9CY62|RN181_MOUSE −0.196052 0.018210542 Tifasp|Q793I8|TIFA_MOUSE 0.20417 0.017490009 Cldn12 sp|Q9ET43|CLD12_MOUSE0.21609 0.013606468 Mansc1 sp|Q9CR33|MANS1_MOUSE 0.2331 0.012257618 Acesp|P09470|ACE_MOUSE 0.23549 0.012272275 Slc32a1 sp|O35633|VIAAT_MOUSE0.23608 0.018205318 Trim26 sp|Q99PN3|TRI26_MOUSE 0.24838 0.010402106Ppp1r2 sp|Q9DCL8|IPP2_MOUSE 0.27392 0.007511401 Plg sp|P20918|PLMN_MOUSE0.29352 0.008522427 Cryba4 sp|Q9JJV0|CRBA4_MOUSE 0.30859 0.005796556Lgals1 sp|P16045|LEG1_MOUSE 0.32733 0.007072827 Enpp3sp|Q6DYE8|ENPP3_MOUSE 0.33051 0.011514242 Pmch sp|P56942|MCH_MOUSE0.36459 0.00464163 Hmgcs2 sp|P54869|HMCS2_MOUSE 0.37081 0.01189837Ppp1r14b sp|Q62084|PP14B_MOUSE 0.37357 0.004635838 Mthfd2sp|P18155|MTDC_MOUSE 0.39935 0.0041522 Hint1 sp|P70349|HINT1_MOUSE0.67922 0.013271695 Hebp1 sp|Q9R257|HEBP1_MOUSE 0.87191 0.017214249Tmem126b sp|Q9D1R1|T126B_MOUSE 1.35616 3.20321E−05

The proteomics data are deposited in the NCBI GEO database underaccession number PXD013989, and is incorporated by reference herein inits entirety.

An assay was undertaken to determine if 2510002D24Rik and Atp23interact. Small proteins such as 2510002D24Rik (105-amino acids)generally show poor immunoreactivity, and antibodies against them arenot effective in immunoprecipitation experiments. Accordingly, theCRISPR/Cas9 approach was used to produce a mutant mouse with2510002D24Rik knocked in and fused with a human influenza hemagglutinin(HA) tag. HA was reliably detected in homozygous knock-in mice (Rik^(HA)mice) but was absent in WT mice (see FIG. 12B). The data of FIG. 2Dshows that Atp23 co-precipitated with HA in Rik^(HA) mice, suggestingthat the 2 proteins are in a complex. Because Atp23 is a mitochondrialintermembrane space protein and is required for the maturation of themitochondrially encoded F₀-subunit Atp6 and its assembly into theF₁F₀-ATP synthase complex²⁰⁻²², an assay was performed to determine if2510002D24Rik is also localized to the mitochondria. HA wassubstantially enriched in the mitochondrial fraction, compared tosynaptosomal fraction from Rik^(HA) hippocampal homogenates, and wasabsent in both WT fractions (as shown in FIG. 2E). These results suggestthat 2510002D24Rik is a mitochondrial protein, consistent with that2510002D24Rik amino acid sequence contains 2 CX₉C domains characteristicof mitochondrial intermembrane space proteins.²³

The interaction between 2510002D24Rik and Atp23 suggested that theseproteins affect ATP equilibrium, which is essential for proper neuralfunction²⁴ and especially important for fast-spiking interneurons thatuse excessive energy.¹³ A recombinant adeno-associated virus (AAV)encoding the fluorescent ATP/ADP sensor PercevalHR²⁵, under control ofthe neuron-specific promoter hSynapsin (AAV-hSyn-PercevalHR), was usedto measure ATP and ADP in individual neurons in hippocampal slices fromadult mice. Several weeks after the injections of this virus into theCA2 area, recordings of fluorescently labelled interneurons or pyramidalneurons were made by using 2-photon imaging and whole-cell current-clamprecordings. A depolarizing current (300 pA, 2 s) injected into a cellbody of labelled fast-spiking interneurons produced an increase in ADP(measured by fluorescence excited at 840 nm) and a decrease in ATP(measured by fluorescence excited at 940 nm). The data is shown in FIG.2F.

Compared to WT CA2 interneurons, Rik^(+/−) and Rik^(−/−) CA2interneurons showed smaller changes in ATP and ADP levels during theevoked train of APs (see FIG. 2H), indicating reduced interconversion ofATP and ADP during the rapid firing in mutant CA2 interneurons. Incomparison, activity-dependent increase in ATP and ADP was notsignificantly different between genotypes in CA2 pyramidal neurons, asshown in FIGS. 13A-13C. Three-dimensional (3D) electron microscopy ofthe CA2 area revealed that approximately 80% of inhibitory (symmetric)synapses contained mitochondria, but only about 20% of excitatory(asymmetric) synapses did. See FIG. 14B. These ratios were not differentbetween the genotypes, suggesting that functional (rather thanstructural) mitochondrial deficits underlie deficient ATP availabilityin CA2 interneurons of 2510002D24Rik-deficient mice.

CRISPR/Cas9-mediated depletion of Atp23 resulted in phenocopying of CA2neuronal, synaptic, and behavioral abnormalities of2510002D24Rik-deficient mice. Atp23^(−/−) mice were embryonicallylethal, and Atp23+/− mice developed normally but showed a reduction ofthe Atp23 transcript by about 50% (see FIG. 15B). Atp23^(+/−) mice hadreduced firing of APs in CA2 interneurons and reduced CA3-CA2 iLTD, asshown in FIGS. 3A and 38. These mutants showed no reduction ininteraction time from a novel to a familiar mouse in the one-chamberdirect-interaction (social recognition) test, whereas their WTlittermates did (as shown in FIG. 3C). Atp23^(+/−) mice performedsimilarly to WT mice during trials 1 and 2, when 2 different novel micewere encountered (as shown in FIG. 3D).

Social memory deficit was also caused by knocking down 2510002D24Rikonly in CA2 interneurons. AAVs expressing cre recombinase (cre) and GFP,under control of the human form of the Dlx5/6 enhancer (hDlx)², whichrestricts expression to GABAergic interneurons, were injected into theCA2 area (CA2^(AVV-hDlx-cre)) of newly developed mutant mice carryingthe floxed 2510002D24Rik alleles (Rik^(f/f) mice). GFP⁺ fast-spikinginterneurons fired fewer APs after depolarization, and iLTD recordedfrom CA2 pyramidal neurons was reduced in Rik^(f/f);CA2^(AVV-hDlx-cre)mice compared to WT;CA2^(AVV-hDlx-cre) mice, as shown in FIGS. 3E and3F. Furthermore, Rik^(f/f);CA2^(AVV-hDlx-cre) mice performed worse thanWT;CA2^(AVV-hDlx-cre) mice in the one-chamber social-recognition testbut not the sociability test. See FIGS. 3G and 3H.

An attempt to rescue neural, synaptic, and social memory abnormalitieswas made by replenishing Atp23 in the CA2 interneurons of2510002D24Rik-deficient mice. AAVs expressing Atp23 fused with GFP orred fluorescent protein (RFP) alone, under control of fsst, apan-GABAergic interneuron promoter²⁷, were injected into the CA2 area(CA2^(AVV-fsst-Atp23) or CA2^(AVV-fsst-RFP)) of WT, Rik^(+/−), andRik^(−/−) mice (as shown in FIG. 4A). Recording from fluorescentlylabelled fast-spiking cells revealed that Atp23 overexpression but notRFP increased firing of APs in CA2 2510002D24Rik-deficient CA2interneurons to the WT levels without affecting firing in WTinterneurons (as shown in FIG. 4B). FIG. 4C shows that CA3-CA2 iLTD wasalso rescued and expressed at WT levels when both mutants were injectedwith CA2^(AVV-fsst-Atp23) but not CA2^(AVV-fsst-RFP). Long-termdisinhibition of CA2 pyramidal neurons was also restored to the WT levelin mutants injected with CA2^(AVV-fsst-Atp23) but not CA2^(AVV-fsst-RFP)(as shown in FIG. 4D). These results suggest that deficient ADP/ATPinterconversion caused by Atp23 depletion underlies neural and synapticabnormalities in 2510002D24Rik-deficient mice. AAV-mediatedoverexpression of ATP23 also rescued the social-recognition deficit (seeFIG. 4E), but a control AAV did not.

2510002D24Rik was identified herein as a major contributor tohippocampal CA2-dependent social memory deficiency associated with22q11DS. Interaction between 2 mitochondrial proteins encoded by nucleargenes 2510002D24Rik and Atp23 underlies reduced firing of APs in CA2fast-spiking interneurons and subsequent reduction in CA3-CA2 iLTD anddisinhibitory drive at CA3-CA2 synapses. This causes deficient socialmemory, presumably due to a reduced output from CA2 to the hypothalamus,ventral CA1 area, and other regions involved in social memory.^(2,28-30)2510002D24Rik deficiency-mediated energy imbalance in fast-spikinginterneurons, which require high energy expenditure¹³ and areparticularly numerous in the CA2 area¹⁴, makes CA2 interneuronsespecially vulnerable. At least 7 other genes that encode mitochondrialproteins within the 22q11.2 region³¹ could exacerbate this metabolicvulnerability and cause interneuron-specific cell death³², as seen inmouse models of 27-gene 22q11DS microdeletion.⁸ Social dysfunction is amajor symptom of many neuropsychiatric and neurologic diseases^(33,34),and the CA2 region plays a crucial role in sociocognitiveprocessing.^(35,33)

Sequences UPF0545 protein isoform 1 encoded by human2510002D24Rik gene (also known as Human gene C22orf39): SEQ ID NO: 1MCRCSLVLLSVDHEVPFSSFFIGWRTEGRAWRAGRPDMADGSGWQPPRPCEAYRAEWKLCRSARHFLHHYYVHGERPACEQWQRDLASCRDWEERRNAEAQQSLCESERARVRAARKHILVWAPRQSPPPDWHLPLPQEKDEprotein encoded by human Atp23 gene SEQ ID NO: 2MAGAPDERRRGPAAGEQLQQQHVSCQVFPERLAQGNPQQGFFSSFFTSNQKCQLRLLKTLETNPYVKLLLDAMKHSGCAVNKDRHFSCEDCNGNVSGGFDASTSQIVLCQNNIHNQAHMNRVVTHELIHAFDHCRAHVDWFTNIRHLACSEVRAANLSGDCSLVNEIFRLHFGLKQHHQTCVRDRATLSILAVRNISKEVAKKAVDEVFESCFNDHEPFGRIPHNKTYARYAHRDFENRDRYYSNIprotein encoded by mouse 2510002D24Rik gene (alsoknown as UPF0545 protein C22orf39 homolog) SEQ ID NO: 3MAVAGSWQPPRPCEVYRAEWELCRSVGHVLHHYYVHGKRPDCRQWLRDLTNCREWEESRSAEAQRSLCESEQVRVQAAQKHTLVWALRQRPPTDWNLPLP QEKDKprotein encoded by mouse Atp23 gene (Uniprot: G3UW46) SEQ ID NO: 4MAGAPGGGELGPAAGEPLLQRPDSGQGSPEPPAHGKPQQGFLSSLFTRDQSCPLMLQKTLDTNPYVKLLLDAMKHSGCAVNRGRHFSCEVCDGNVSGGFDASTSQIVLCENNIRNQAHMGRVVTHELIHAFDHCRAHVHWFTNIRHLACSEIRAASLSGDCSLVNELFRLRFGLKQHHQTCVRDRAVLSILAVRNVSREEAQKAVDEVFQTCFNDREPFGRIPHNQTYARYAHRDFQNRDRYYSNIprotein encoded by mouse Atp23 gene (Uniprot: Q9CWQ3) SEQ ID NO: 5MAGAPGGGELGPAAGEPLLQRPDSGQGSPEPPAHGKPQQGFLSSLFTRDQSCPLMLQKTLDTNPYVKLLLDAMKHSGCAVNRGRHFSCEVCDGNVSGGFDASTSQIVLCENNIRNQAHMGRVVTHELIHAFDHCRAHVHWFTNIRHLACSEIRAASLSGDCSLVNELFRLRFGLKQHHQIETSCVSRPAMNSQSCLGLVS A

REFERENCES

-   1. Hitti, F. L. & Siegelbaum, S. A. The hippocampal CA2 region is    essential for social memory. Nature 508, 88-92 (2014).-   2. Meira, T. et al. A hippocampal circuit linking dorsal CA2 to    ventral CA1 critical for social memory dynamics. Nat. Commun. 9,    4163 (2018).-   3. Chevaleyre, V. & Piskorowski, R. A. Hippocampal Area CA2: An    Overlooked but Promising Therapeutic Target. Trends Mol Med. 22,    645-655 (2016).-   4. Smith, A. S., Williams Avram, S. K., Cymerblit-Sabba, A.,    Song, J. & Young, W. S. Targeted activation of the hippocampal CA2    area strongly enhances social memory. Mol Psychiatry 21, 1137-1144    (2016).-   5. Stevenson, E. L. & Caldwell, H. K. Lesions to the CA2 region of    the hippocampus impair social memory in mice. Eur. J Neurosci 40,    3294-3301 (2014).-   6. Dudek, S. M., Alexander, G. M. & Farris, S. Rediscovering area    CA2: unique properties and functions. Nat. Rev. Neurosci. 17, 89-102    (2016).-   7. Piskorowski, R. A. & Chevaleyre, V. Memory circuits: CA2. Curr.    Opin. Neurobiol. 52, 54-59 (2018).-   8. Piskorowski, R. A. et al. Age-dependent specific changes in area    CA2 of the hippocampus and social memory deficit in a mouse model of    the 22q11.2 deletion syndrome. Neuron 89, 163-176 (2016).-   9. Scambler, P. J. et al. Velo-cardio-facial syndrome associated    with chromosome 22 deletions encompassing the DiGeorge locus. Lancet    339, 1138-1139 (1992).-   10. Karayiorgou, M., Simon, T. J. & Gogos, J. A. 22q11.2    microdeletions: linking DNA structural variation to brain    dysfunction and schizophrenia. Nat Rev Neurosci 11, 402-416 (2010).-   11. McDonald-McGinn, D. M. et al. 22q11.2 deletion syndrome. Nat Rev    Dis Prim. 1, 15071 (2015).-   12. Kragness, S., Harrison, M. A. A., Westmoreland, J. J.,    Burstain, A. & Earls, L. R. Age-dependent expression pattern in the    mammalian brain of a novel, small peptide encoded in the 22q11.2    deletion syndrome region. Gene Expr. Patterns 28, 95-103 (2018).-   13. Kann, O. The interneuron energy hypothesis: Implications for    brain disease. Neurobiol. Dis. 90, 75-85 (2016).-   14. Botcher, N. A., Falck, J. E., Thomson, A. M. & Mercer, A.    Distribution of interneurons in the CA2 region of the rat    hippocampus. Front. Neuroanat. 8, (2014).-   15. DeVito, L. M. et al. Vasopressin 1b Receptor Knock-Out Impairs    Memory for Temporal Order. J. Neurosci. 29, 2676-2683 (2009).-   16. Brennan, P. A. & Zufall, F. Pheromonal communication in    vertebrates. Nature 444, 308-315 (2006).-   17. Stark, K. L. et al. Altered brain microRNA biogenesis    contributes to phenotypic deficits in a 22q11-deletion mouse model.    Nat Genet 40, 751-760 (2008).-   18. Zhao, M., Choi, Y.-S., Obrietan, K. & Dudek, S. M. Synaptic    Plasticity (and the Lack Thereof) in Hippocampal CA2 Neurons. J.    Neurosci. 27, 12025-12032 (2007).-   19. Nasrallah, K., Piskorowski, R. A. & Chevaleyre, V. Inhibitory    Plasticity Permits the Recruitment of CA2 Pyramidal Neurons by    CA3(1,2,3). eNeuro. 2, (2015).-   20. Osman, C., Wilmes, C., Tatsuta, T. & Langer, T. Prohibitins    Interact Genetically with Atp23, a Novel Processing Peptidase and    Chaperone for the F1FO-ATP Synthase. Mol. Biol. Cell 18, 627-635    (2007).-   21. Weckbecker, D., Longen, S., Riemer, J. & Herrmann, J. M. Atp23    biogenesis reveals a chaperone-like folding activity of Mia40 in the    IMS of mitochondria. EMBO J. 31, 4348-4358 (2012).-   22. Zeng, X., Neupert, W. & Tzagoloff, A. The metalloprotease    encoded by ATP23 has a dual function in processing and assembly of    subunit 6 of mitochondrial ATPase. Mol Biol Cell 18, 617-626 (2007).-   23. Modjtahedi, N., Tokatlidis, K., Dessen, P. & Kroemer, G.    Mitochondrial Proteins Containing    Coiled-Coil-Helix-Coiled-Coil-Helix (CHCH) Domains in Health and    Disease. Trends Biochem. 41, 245-260 (2016).-   24. Rajendran, M., Dane, E., Conley, J. & Tantama, M. Imaging    Adenosine Triphosphate (ATP). Biol. Bull. 231, 73-84 (2016).-   25. Tantama, M., Martinez-Frangois, J. R., Mongeon, R. & Yellen, G.    Imaging energy status in live cells with a fluorescent biosensor of    the intracellular ATP-to-ADP ratio. Nat Commun 4, (2013).-   26. Dimidschstein, J. et al. A viral strategy for targeting and    manipulating interneurons across vertebrate species. Nat Neurosci    19, 1743-1749 (2016).-   27. Nathanson, J. L. et al. Short Promoters in Viral Vectors Drive    Selective Expression in Mammalian Inhibitory Neurons, but do not    Restrict Activity to Specific Inhibitory Cell-Types. Front Neural    Circuits. 3, 19 (2009).-   28. Okuyama, T., Kitamura, T., Roy, D. S., Itohara, S. &    Tonegawa, S. Ventral CA1 neurons store social memory. Science    (80-.). 353, 1536-1541 (2016).-   29. Cui, Z., Gerfen, C. R. & Young, W. S. Hypothalamic and other    connections with dorsal CA2 area of the mouse hippocampus. J. Comp.    Neurol. 521, 1844-1866 (2013).-   30. Rowland, D. C. et al. Transgenically Targeted Rabies Virus    Demonstrates a Major Monosynaptic Projection from Hippocampal Area    CA2 to Medial Entorhinal Layer II Neurons. J. Neurosci. 33,    14889-14898 (2013).-   31. Devaraju, P. & Zakharenko, S. S. Mitochondria in complex    psychiatric disorders: Lessons from mouse models of 22q11.2 deletion    syndrome. Bioessays 39, (2017).-   32. Harris, J. J., Jolivet, R. & Attwell, D. Synaptic Energy Use and    Supply. Neuron 75, 762-777 (2012).-   33. Chevallier, C., Kohls, G., Troiani, V., Brodkin, E. S. &    Schultz, R. T. The social motivation theory of autism. Trends Cogn.    Sci. 16, 231-239 (2012).-   34. Green, M. F., Horan, W. P. & Lee, J. Social cognition in    schizophrenia. Nat Rev Neurosci 16, 620-631 (2015).-   35. Piskorowski, R. A. & Chevaleyre, V. Memory circuits: CA2. Curr.    Opin. Neurobiol. 52, 54-59 (2018).-   36. Carstens, K. E. & Dudek, S. M. Regulation of synaptic plasticity    in hippocampal area CA2. Curr. Opin. Neurobiol. 54, 194-199 (2019).-   37. Moy, S. S. et al. Sociability and preference for social novelty    in five inbred strains: an approach to assess autistic-like behavior    in mice. Genes, Brain Behav. 3, 287-302 (2004).-   38. Yang, M. & Crawley, J. N. Simple behavioral assessment of mouse    olfaction.

Curr. Protoc. Neurosci. Chapter 8, Unit 8.24 (2009).

-   39. Sims, N. R. & Anderson, M. F. Isolation of mitochondria from rat    brain using Percoll density gradient centrifugation. Nat. Protoc. 3,    1228-1239 (2008).-   40. Kristian, T. Isolation of Mitochondria from the CNS. in Current    Protocols in Neuroscience (John Wiley & Sons, Inc., 2001).    doi:10.1002/0471142301.ns0722s52-   41. Xu, P., Duong, D. M. & Peng, J. Systematical Optimization of    Reverse-Phase Chromatography for Shotgun Proteomics. J. Proteome    Res. 8, 3944-3950 (2009).-   42. Pagala, V. R. et al. Quantitative Protein Analysis by Mass    Spectrometry. in Protein-Protein Interactions: Methods and    Applications (eds. Meyerkord, C. L. & Fu, H.) 281-305 (Springer New    York, 2015). doi:10.1007/978-1-4939-2425-7_17-   43. Wang, X. et al. JUMP: A Tag-based Database Search Tool for    Peptide Identification with High Sensitivity and Accuracy. Mol.    Cell. Proteomics 13, 3663-3673 (2014).-   44. Denk, W. & Horstmann, H. Serial block-face scanning electron    microscopy to reconstruct three-dimensional tissue nanostructure.    PLoS. Biol. 2, e329 (2004).

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

All patents, applications, publications, test methods, literature, andother materials cited herein are hereby incorporated by reference intheir entirety as if physically present in this specification.

1. A method for treating a social memory deficit in a subject with aneuropsychiatric disease, said method comprising administering to thesubject a therapeutically effective amount of (i) a protein encoded by a2510002D24Rik gene or a functional derivative or fragment thereof, (ii)a vector expressing a protein encoded by a 2510002D24Rik gene or afunctional derivative or fragment thereof, (iii) a protein encoded by anAtp23 gene or a functional derivative or fragment thereof, or (iv) avector expressing a protein encoded by a Atp23 gene or a functionalderivative or fragment thereof. 2-6. (canceled)
 7. The method of claim1, wherein the neuropsychiatric disease is selected from schizophreniaspectrum disorders, 22q11 deletion syndrome, and autism spectrumdisorders.
 8. The method of claim 1, wherein the vector is selected fromadeno-associated virus (AAV) vectors, retrovirus vectors, adenovirusvectors, Sindbis virus vectors, vaccinia virus vectors, and herpes virusvectors.
 9. The method of claim 8, wherein the vector is an AAV vector.10-11. (canceled)
 12. The method of claim 1, wherein the expression ofthe protein or functional derivative or fragment thereof is controlledby a promoter selected from a pan-GABAergic interneuron promoter, fsstpromoter, hDlx promoter, mDlx promoter, Synapsin promoter, CMV promoter,β-actin promoter, and CamKIIa promoter.
 13. (canceled)
 14. The method ofclaim 1, wherein the administration is via injection into the CA2 areaof the hippocampus of the subject.
 15. The method of claim 1, whereinthe administration is via a transcranial surgical injection.
 16. Themethod of claim 1, wherein the administration is systemic.
 17. Themethod of claim 1, wherein the administration is intranasal.
 18. Themethod of claim 1, wherein the protein encoded by said 2510002D24Rikgene comprises the amino acid sequence which has at least 80% sequenceidentity to SEQ ID NO:
 1. 19. The method of claim 18, wherein theprotein encoded by said 2510002D24Rik gene comprises the amino acidsequence which has at least 90% sequence identity to SEQ ID NO:
 1. 20.The method of claim 19, wherein the protein encoded by said2510002D24Rik gene comprises the amino acid sequence SEQ ID NO:
 1. 21.The method of claim 20, wherein the protein encoded by said2510002D24Rik gene consists of the amino acid sequence SEQ ID NO:
 1. 22.The method of claim 1, wherein the protein encoded by said Atp23 genecomprises the amino acid sequence which has at least 80% sequenceidentity to SEQ ID NO:
 2. 23. The method of claim 22, wherein theprotein encoded by said Atp23 gene comprises the amino acid sequencewhich has at least 90% sequence identity to SEQ ID NO:
 2. 24. The methodof claim 23, wherein the protein encoded by said Atp23 gene comprisesthe amino acid sequence SEQ ID NO:
 2. 25. The method of claim 24,wherein the protein encoded by said Atp23 gene consists of the aminoacid sequence SEQ ID NO:
 2. 26. The method of claim 1, wherein thesubject is human.
 27. A pharmaceutical composition comprising (i) aprotein encoded by a 2510002D24Rik gene or a functional derivative orfragment thereof and a pharmaceutically acceptable carrier or excipient,or (ii) a vector encoding a protein encoded by a 2510002D24Rik gene or afunctional derivative or fragment thereof and a pharmaceuticallyacceptable carrier or excipient. 28-37. (canceled)
 38. A pharmaceuticalcomposition comprising (i) a protein encoded by said Atp23 gene or afunctional derivative or fragment thereof and a pharmaceuticallyacceptable carrier or excipient, or (ii) a vector encoding a proteinencoded by said Atp23 gene or a functional derivative or fragmentthereof and a pharmaceutically acceptable carrier or excipient. 39-50.(canceled)